High-Throughput Screening to Advance In Vitro Toxicology: Accomplishments, Challenges, and Future Directions.

Traditionally, chemical toxicity is determined by in vivo animal studies, which are low throughput, expensive, and sometimes fail to predict compound toxicity in humans. Due to the increasing number of chemicals in use and the high rate of drug candidate failure due to toxicity, it is imperative to develop in vitro, high-throughput screening methods to determine toxicity. The Tox21 program, a unique research consortium of federal public health agencies, was established to address and identify toxicity concerns in a high-throughput, concentration-responsive manner using a battery of in vitro assays. In this article, we review the advancements in high-throughput robotic screening methodology and informatics processes to enable the generation of toxicological data, and their impact on the field; further, we discuss the future of assessing environmental toxicity utilizing efficient and scalable methods that better represent the corresponding biological and toxicodynamic processes in humans.

Protecting Human and Animal Health: The Road from Animal Models to New Approach Methods.

Animals and animal models have been invaluable for our current understanding of human and animal biology, including physiology, pharmacology, biochemistry, and disease pathology. However, there are increasing concerns with continued use of animals in basic biomedical, pharmacological, and regulatory research to provide safety assessments for drugs and chemicals. There are concerns that animals do not provide sufficient information on toxicity and/or efficacy to protect the target population, so scientists are utilizing the principles of replacement, reduction, and refinement (the 3Rs) and increasing the development and application of new approach methods (NAMs). NAMs are any technology, methodology, approach, or assay used to understand the effects and mechanisms of drugs or chemicals, with specific focus on applying the 3Rs. Although progress has been made in several areas with NAMs, complete replacement of animal models with NAMs is not yet attainable. The road to NAMs requires additional development, increased use, and, for regulatory decision making, usually formal validation. Moreover, it is likely that replacement of animal models with NAMs will require multiple assays to ensure sufficient biologic coverage. The purpose of this manuscript is to provide a balanced view of the current state of the use of animal models and NAMs as approaches to development, safety, efficacy, and toxicity testing of drugs and chemicals. Animals do not provide all needed information nor do NAMs, but each can elucidate key pieces of the puzzle of human and animal biology and contribute to the goal of protecting human and animal health. SIGNIFICANCE STATEMENT: Data from traditional animal studies have predominantly been used to inform human health safety and efficacy. Although it is unlikely that all animal studies will be able to be replaced, with the continued advancement in new approach methods (NAMs), it is possible that sometime in the future, NAMs will likely be an important component by which the discovery, efficacy, and toxicity testing of drugs and chemicals is conducted and regulatory decisions are made.

Retrospective analysis of dog study data from food and color additive petitions.

As part of the US FDA CFSAN's efforts to explore alternatives to animal testing, we retrospectively analyzed a sample of food additive (FAP) and color additive petitions (CAP) submitted to the FDA for the utility of dog study data in safety assessment. FAPs and CAPs containing dog studies (161 petitions) were classified as decisive (38%), supportive (27%), supplemental (29%) or undermined (6%) based on the impact the dog study data had on the final safety decision. Petitions classified as decisive were further categorized based on if the dog study data were used to a) address a safety concern (35/61); b) calculate an acceptable daily intake (ADI) (11/61); c) withdraw a petition (4/61); d) the effect was unique to the dog (2/61); or e) unclear (9/61). Of 11 petitions where the dog study was used to set an ADI, 7 contained studies where the points of departure (POD) from the dog studies were within an 8-fold range of the rodent with differences in study design likely contributing to the difference in PODs. Future research should include the development and use of qualified alternative studies to replace the use of animal testing for food and color additive safety assessment while ensuring human safety.

Reassessment of the cadmium toxicological reference value for use in human health assessments of foods.

The U.S. Food and Drug Administration (FDA) developed an oral toxicological reference value (TRV) for characterizing potential health concerns from dietary exposure to cadmium (Cd). The development of the TRV leveraged the FDA's previously published research including (1) a systematic review for adverse health effects associated with oral Cd exposure and (2) a human physiological based pharmacokinetic (PBPK) model adapted from Kjellstrom and Nordberg (1978) for use in reverse dosimetry applied to the U.S. population. Adverse effects of Cd on the bone and kidney are associated with similar points of departure (PODs) of approximately 0.50 µg Cd/g creatinine for females aged 50-60 based on available epidemiologic data. We also used the upper bound estimate of the renal cortical concentration (50 µg/g Cd) occurring in the U.S. population at 50 years of age as a POD. Based on the output from our reverse dosimetry PBPK Model, a range of 0.21-0.36 µg/kg bw/day was developed for the TRV. The animal data used for the animal TRV derivation (0.63-1.8 µg/kg bw/day) confirms biological plausibility for both the bone and kidney endpoints.

Gene expression analyses reveal potential mechanism of inorganic arsenic-induced apoptosis in zebrafish.

Our previous study showed that sodium arsenite (200 mg/L) affected the nervous system and induced motor neuron development via the Sonic hedgehog pathway in zebrafish larvae. To gain more insight into the effects of arsenite on other signaling pathways, including apoptosis, we have performed quantitative polymerase chain reaction array-based gene expression analyses. The 96-well array plates contained primers for 84 genes representing 10 signaling pathways that regulate several biological functions, including apoptosis. We exposed eggs at 5 h postfertilization until the 72 h postfertilization larval stage to 200 mg/L sodium arsenite. In the Janus kinase/signal transducers and activators of transcription, nuclear factor κ-light-chain-enhancer of activated B cells, and Wingless/Int-1 signaling pathways, the expression of only one gene in each pathway was significantly altered. The expression of multiple genes was altered in the p53 and oxidative stress pathways. Sodium arsenite induced excessive apoptosis in the larvae. This compelled us to analyze specific genes in the p53 pathway, including cdkn1a, gadd45aa, and gadd45ba. Our data suggest that the p53 pathway is likely responsible for sodium arsenite-induced apoptosis. In addition, sodium arsenite significantly reduced global DNA methylation in the zebrafish larvae, which may indicate that epigenetic factors could be dysregulated after arsenic exposure. Together, these data elucidate potential mechanisms of arsenic toxicity that could improve understanding of arsenic's effects on human health.

The Tox21 10K Compound Library: Collaborative Chemistry Advancing Toxicology.

Since 2009, the Tox21 project has screened ∼8500 chemicals in more than 70 high-throughput assays, generating upward of 100 million data points, with all data publicly available through partner websites at the United States Environmental Protection Agency (EPA), National Center for Advancing Translational Sciences (NCATS), and National Toxicology Program (NTP). Underpinning this public effort is the largest compound library ever constructed specifically for improving understanding of the chemical basis of toxicity across research and regulatory domains. Each Tox21 federal partner brought specialized resources and capabilities to the partnership, including three approximately equal-sized compound libraries. All Tox21 data generated to date have resulted from a confluence of ideas, technologies, and expertise used to design, screen, and analyze the Tox21 10K library. The different programmatic objectives of the partners led to three distinct, overlapping compound libraries that, when combined, not only covered a diversity of chemical structures, use-categories, and properties but also incorporated many types of compound replicates. The history of development of the Tox21 "10K" chemical library and data workflows implemented to ensure quality chemical annotations and allow for various reproducibility assessments are described. Cheminformatics profiling demonstrates how the three partner libraries complement one another to expand the reach of each individual library, as reflected in coverage of regulatory lists, predicted toxicity end points, and physicochemical properties. ToxPrint chemotypes (CTs) and enrichment approaches further demonstrate how the combined partner libraries amplify structure-activity patterns that would otherwise not be detected. Finally, CT enrichments are used to probe global patterns of activity in combined ToxCast and Tox21 activity data sets relative to test-set size and chemical versus biological end point diversity, illustrating the power of CT approaches to discern patterns in chemical-activity data sets. These results support a central premise of the Tox21 program: A collaborative merging of programmatically distinct compound libraries would yield greater rewards than could be achieved separately.

The ACE Index: mapping childhood adversity in England.

BACKGROUND: Studies of adults show that adverse childhood experiences (ACEs) are associated with health and social problems and are more common among people living in deprived areas. However, there is limited information about the geographical pattern of contemporary ACEs. METHODS: We used data from the police, social services, schools and vital statistics in England to calculate population rates of events that represent childhood adversity. We constructed an 'ACE Index' that summarizes the relative frequency of ACEs at local authority level, informed by the methods of the Index of Multiple Deprivation. We explored associations between the ACE Index and local characteristics in cross-sectional ecological analysis. RESULTS: The ACE Index was strongly associated with the proportion of children that live in income-deprived households (child poverty). In addition, the ACE Index was independently associated with higher population density and was higher in certain regions, particularly the north-east. CONCLUSIONS: The association between ACEs and child poverty provides evidence of a process in which deprivation increases the risk of adverse experiences in childhood. The ACE Index can inform allocation of resources for prevention and mitigation of ACEs.

State of the science on alternatives to animal testing and integration of testing strategies for food safety assessments: Workshop proceedings.

Rapidly evolving technological methods and mechanistic toxicological understanding have paved the way for new science-based approaches for the determination of chemical safety in support of advancing public health. Approaches including read-across, high-throughput screening, in silico models, and organ-on-a-chip technologies were addressed in a 2017 workshop focusing on how scientists can move effectively toward a vision for 21st century food safety assessments. The application of these alternative methods, the need for further development of standardized practices, and the interpretation and communication of results were addressed. Expert presentations encompassed regulatory, industry, and academic perspectives, and the workshop culminated in a panel discussion in which participants engaged experts about current issues pertaining to the application of alternative methods in toxicological testing for food safety assessments.

A review of the evidence to support interim reference level for dietary lead exposure in adults.

FDA developed the interim reference level (IRL) for lead of 3 µg/day in children and 12.5 µg/day in women of childbearing age (WOCBA) to better protect the fetus from lead toxicity. These IRLs correspond to a blood lead level (BLL) of 0.5 µg/dL in both populations. The current investigation was performed to determine if the IRL for WOCBA should apply to the general population of adults. A literature review of epidemiological studies was conducted to determine whether a BLL of 0.5 µg/dL is associated with adverse effects in adults. Some studies reported adverse effects over a wide range of BLLs that included 0.5 µg/dL adding uncertainty to conclusions about effects at 0.5 µg/dL; however, no studies clearly identified this BLL as an adverse effect level. Results also showed that the previously developed PTTDI for adults of 75 µg/day lead may not be health protective, supporting use of a lower reference value for lead toxicity in this population group. Use of the 12.5 µg/day IRL as a benchmark for dietary lead intake is one way FDA will ensure that dietary lead intake in adults is reduced.

U.S. Food and Drug Administration's interim reference levels for dietary lead exposure in children and women of childbearing age.

Reducing lead exposure is a public health priority for the US Food and Drug Administration as well as other federal agencies. The goals of this research were to 1) update the maximum daily dietary intake of lead from food, termed an interim reference level (IRL), for children and for women of childbearing age (WOCBA) and 2) to confirm through a literature review that with the exception of neurodevelopment, which was not evaluated here, no adverse effects of lead consistently occur at the blood lead level (BLL) associated with the IRL. Because no safe level of lead exposure has yet been identified for children's health, the IRLs of 3 µg/day for children and 12.5 µg/day for WOCBA were derived from the Centers for Disease Control and Prevention reference value of 5 µg/dL BLL, the level at which public health actions should be initiated. The literature review showed that no adverse effects of lead consistently occurred at the BLL associated with the IRLs (0.5 µg/dL). The IRLs of 3 µg/day for children and 12.5 µg/day for WOCBA should serve as useful benchmarks in evaluating the potential for adverse effects of dietary lead.

An evaluation framework for new approach methodologies (NAMs) for human health safety assessment.

The need to develop new tools and increase capacity to test pharmaceuticals and other chemicals for potential adverse impacts on human health and the environment is an active area of development. Much of this activity was sparked by two reports from the US National Research Council (NRC) of the National Academies of Sciences, Toxicity Testing in the Twenty-first Century: A Vision and a Strategy (2007) and Science and Decisions: Advancing Risk Assessment (2009), both of which advocated for "science-informed decision-making" in the field of human health risk assessment. The response to these challenges for a "paradigm shift" toward using new approach methodologies (NAMS) for safety assessment has resulted in an explosion of initiatives by numerous organizations, but, for the most part, these have been carried out independently and are not coordinated in any meaningful way. To help remedy this situation, a framework that presents a consistent set of criteria, universal across initiatives, to evaluate a NAM's fit-for-purpose was developed by a multi-stakeholder group of industry, academic, and regulatory experts. The goal of this framework is to support greater consistency across existing and future initiatives by providing a structure to collect relevant information to build confidence that will accelerate, facilitate and encourage development of new NAMs that can ultimately be used within the appropriate regulatory contexts. In addition, this framework provides a systematic approach to evaluate the currently-available NAMs and determine their suitability for potential regulatory application. This 3-step evaluation framework along with the demonstrated application with case studies, will help build confidence in the scientific understanding of these methods and their value for chemical assessment and regulatory decision-making.

What works in inclusion health: overview of effective interventions for marginalised and excluded populations.

Inclusion health is a service, research, and policy agenda that aims to prevent and redress health and social inequities among the most vulnerable and excluded populations. We did an evidence synthesis of health and social interventions for inclusion health target populations, including people with experiences of homelessness, drug use, imprisonment, and sex work. These populations often have multiple overlapping risk factors and extreme levels of morbidity and mortality. We identified numerous interventions to improve physical and mental health, and substance use; however, evidence is scarce for structural interventions, including housing, employment, and legal support that can prevent exclusion and promote recovery. Dedicated resources and better collaboration with the affected populations are needed to realise the benefits of existing interventions. Research must inform the benefits of early intervention and implementation of policies to address the upstream causes of exclusion, such as adverse childhood experiences and poverty.

Exploring current read-across applications and needs among selected U.S. Federal Agencies.

Read-across is a well-established data gap-filling technique applied for regulatory purposes. In US Environmental Protection Agency's New Chemicals Program under TSCA, read-across has been used extensively for decades, however the extent of application and acceptance of read-across among U.S. federal agencies is less clear. In an effort to build read-across capacity, raise awareness of the state of the science, and work towards a harmonization of read-across approaches across U.S. agencies, a new read-across workgroup was established under the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). This is one of several ad hoc groups ICCVAM has convened to implement the ICCVAM Strategic Roadmap. In this article, we outline the charge and scope of the workgroup and summarize the current applications, tools used, and needs of the agencies represented on the workgroup for read-across. Of the agencies surveyed, the Environmental Protection Agency had the greatest experience in using read-across whereas other agencies indicated that they would benefit from gaining a perspective of the landscape of the tools and available guidance. Two practical case studies are also described to illustrate how the read-across approaches applied by two agencies vary on account of decision context.

Status of acute systemic toxicity testing requirements and data uses by U.S. regulatory agencies.

Acute systemic toxicity data are used by a number of U.S. federal agencies, most commonly for hazard classification and labeling and/or risk assessment for acute chemical exposures. To identify opportunities for the implementation of non-animal approaches to produce these data, the regulatory needs and uses for acute systemic toxicity information must first be clarified. Thus, we reviewed acute systemic toxicity testing requirements for six U.S. agencies (Consumer Product Safety Commission, Department of Defense, Department of Transportation, Environmental Protection Agency, Food and Drug Administration, Occupational Safety and Health Administration) and noted whether there is flexibility in satisfying data needs with methods that replace or reduce animal use. Understanding the current regulatory use and acceptance of non-animal data is a necessary starting point for future method development, optimization, and validation efforts. The current review will inform the development of a national strategy and roadmap for implementing non-animal approaches to assess potential hazards associated with acute exposures to industrial chemicals and medical products. The Acute Toxicity Workgroup of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), U.S. agencies, non-governmental organizations, and other stakeholders will work to execute this strategy.

In vitro exposure systems and dosimetry assessment tools for inhaled tobacco products: Workshop proceedings, conclusions and paths forward for in vitro model use.

In 2009, the passing of the Family Smoking Prevention and Tobacco Control Act facilitated the establishment of the FDA Center for Tobacco Products (CTP), and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed 'modified risk'. On 4-6 April 2016, the Institute for In Vitro Sciences, Inc. (IIVS) convened a workshop conference entitled, In Vitro Exposure Systems and Dosimetry Assessment Tools for Inhaled Tobacco Products, to bring together stakeholders representing regulatory agencies, academia and industry to address the research priorities articulated by the FDA CTP. Specific topics were covered to assess the status of current in vitro smoke and aerosol/vapour exposure systems, as well as the various approaches and challenges to quantifying the complex exposures in in vitro pulmonary models developed for evaluating adverse pulmonary events resulting from tobacco product exposures. The four core topics covered were: a) Tobacco Smoke and E-Cigarette Aerosols; b) Air-Liquid Interface-In Vitro Exposure Systems; c) Dosimetry Approaches for Particles and Vapours/In Vitro Dosimetry Determinations; and d) Exposure Microenvironment/Physiology of Cells. The 2.5-day workshop included presentations from 20 expert speakers, poster sessions, networking discussions, and breakout sessions which identified key findings and provided recommendations to advance these technologies. Here, we will report on the proceedings, recommendations, and outcome of the April 2016 technical workshop, including paths forward for developing and validating non-animal test methods for tobacco product smoke and next generation tobacco product aerosol/vapour exposures. With the recent FDA publication of the final deeming rule for the governance of tobacco products, there is an unprecedented necessity to evaluate a very large number of tobacco-based products and ingredients. The questionable relevance, high cost, and ethical considerations for the use of in vivo testing methods highlight the necessity of robust in vitro approaches to elucidate tobacco-based exposures and how they may lead to pulmonary diseases that contribute to lung exposure-induced mortality worldwide.

Trust your gut: Establishing confidence in gastrointestinal models - An overview of the state of the science and contexts of use.

The webinar series and workshop titled "Trust Your Gut: Establishing Confidence in Gastrointestinal Models ­ An Overview of the State of the Science and Contexts of Use" was co-organized by NICEATM, NIEHS, FDA, EPA, CPSC, DoD, and the Johns Hopkins Center for Alternatives to Animal Testing (CAAT) and hosted at the National Institutes of Health in Bethesda, MD, USA on October 11-12, 2023. New approach methods (NAMs) for assessing issues of gastrointestinal tract (GIT)- related toxicity offer promise in addressing some of the limitations associated with animal-based assessments. GIT NAMs vary in complexity, from two-dimensional monolayer cell line-based systems to sophisticated 3-dimensional organoid systems derived from human primary cells. Despite advances in GIT NAMs, challenges remain in fully replicating the complex interactions and pro­cesses occurring within the human GIT. Presentations and discussions addressed regulatory needs, challenges, and innovations in incorporating NAMs into risk assessment frameworks; explored the state of the science in using NAMs for evaluating systemic toxicity, understanding absorption and pharmacokinetics, evaluating GIT toxicity, and assessing potential allergenicity; and discussed strengths, limitations, and data gaps of GIT NAMs as well as steps needed to establish confidence in these models for use in the regulatory setting.

Non-animal methods to assess whether chemicals may be toxic to the human digestive tract promise to complement or improve on animal-based methods. These approaches, which are based on human or animal cells and/or computer models, are faced with their own technical challenges and need to be shown to predict adverse effects in humans. Regulators are tasked with evaluating submitted data to best protect human health and the environment. A webinar series and workshop brought together scientists from academia, industry, military, and regulatory authorities from dif­ferent countries to discuss how non-animal methods can be integrated into the risk assessment of drugs, food additives, dietary supplements, pesticides, and industrial chemicals for gastrointestinal toxicity.

PROTOCOL: An evidence and gap map of studies of implementation issues for interventions for those affected by and at risk of homelessness in high-income countries.

This is the protocol for a Campbell systematic review. The objectives are as follows. The proposed evidence and gap map will present relevant process evaluations and other studies of barriers and facilitators, both qualitative and quantitative, for eligible homelessness interventions to highlight the issues arising in the implementation of these interventions. Specifically, the objectives of the map are to: (i) develop a clear taxonomy of interventions and implementation issues (e.g., barriers and facilitators-factors which works as barriers to hinder successful implementation of policies and programmes and factors which facilitate the intervention and therefore support its implementation) related to homelessness in high-income countries; (ii) map available systematic reviews and primary studies of the implementation issues of interventions for those experiencing homelessness and those at risk of homelessness, with an overview provided in a summary report; (iii) provide a searchable database of included studies accessible to research users via CHI website.

Inorganic arsenic alters the development of dopaminergic neurons but not serotonergic neurons and induces motor neuron development via Sonic hedgehog pathway in zebrafish.

The mechanism of inorganic arsenic-induced neurotoxicity at the cellular level is not known. In zebrafish, teratological effects of inorganic arsenic have been shown at various concentrations. Here, we used similar concentrations of inorganic arsenic to evaluate the effects on specific neuron types. Exposure of zebrafish embryos at 5 h post fertilization (hpf) to sodium arsenite induced developmental toxicity (reduced body length) in 72 hpf larvae, beginning at a concentration of 300 mg/L concentration. Mortality or overt morphological deformity was detected at 500 mg/L sodium arsenite. While 200 mg/L sodium arsenite induced development of tyrosine hydroxylase-positive (dopaminergic) neurons, there was no significant effect on the development of 5-hydroxytryptamine (serotonergic) neurons. Sodium arsenite reduced acetylcholinesterase activity. In the hb9-GFP transgenic larvae, both 200 and 400 mg/L sodium arsenite produced supernumerary motor neurons in the spinal cord. Inhibition of the Sonic hedgehog (Shh) pathway that is essential for motor neuron development, by Gant61, prevented sodium arsenite-induced supernumerary motor neuron development. Inductively coupled plasma mass spectrometry (ICP-MS) revealed that with 200 mg/L and 400 mg/L sodium arsenite treatment, each larva had an average of 387.8 pg and 847.5 pg arsenic, respectively. The data show for the first time that inorganic arsenic alters the development of dopaminergic and motor neurons in the zebrafish larvae and the latter occurs through the Shh pathway. These results may help understand why arsenic-exposed populations suffer from psychiatric disorders and motor neuron disease and Shh may, potentially, serve as a plasma biomarker of arsenic toxicity.