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ICCVAM member agencies work to promote the regulatory acceptance of new, scientifically valid toxicological tests that protect human and animal health and the environment while replacing, reducing, or refining animal tests. To achieve this goal, many ICCVAM member agencies engage in research activities that focus both on developing new test methods and exploring new technologies that may support future test method development. Effective translation of technological advances into new test methods should allow better protection of public health while addressing animal use and welfare concerns.
The interagency Tox21 research initiative uses in vitro, high-throughput screening (HTS) assays to test a broad variety of substances and considers data from the screenings collectively to assess effects on biological pathways related to toxicity.
The tenth anniversary of Tox21 was observed in 2017. The program’s accomplishments during that time included use of knowledge gained through Tox21 to inform policy and regulatory decisions about chemical safety; the publication of more than 200 peer-reviewed articles; and the public availability of millions of data points for research and analysis.
As Tox21 enters its second decade, its new strategic plan features five areas of focus:
Tox21 sponsored the Transform Tox Testing Challenge, which recruited innovative thinkers to find new ways to incorporate chemical metabolism into HTS assays. The goal was to help researchers more accurately assess effects of chemicals and better protect human health.
The first stage of the challenge, launched in January 2016, culminated in a July 2016 workshop, which brought together the 10 Stage 1 challenge winners and other experts to discuss the Tox21 and ToxCast programs, the Stage 1 proposals, and feasible expectations for the remainder of the challenge.
The goal of Stage 2 of the challenge was to encourage the Stage 1 winners to further develop their proposals into practical designs for new chemical screening technologies. Stage 2 winners were announced in November 2017. A third stage of the challenge, which would promote commercial development of the technologies, is currently under consideration.
The U.S. Army continues to explore approaches to reducing animal use in development of medical countermeasures to chemical and biological threats. The Absorption, Distribution, Metabolism, Elimination, and Toxicology Center of Excellence (ADMET CoE) has instituted best practices from the pharmaceutical industry to address this goal. Novel chemical compounds identified as potential therapeutics in target-specific high-throughput and virtual assays are characterized using validated in silico and in vitro assays to predict potential to become a FDA-approved drug. These cost-effective assays identify compounds with unfavorable properties, which are eliminated prior to animal testing. Assays to predict plasma stability, microsomal stability, cytochrome p450 inhibition (drug-drug interaction), intestinal and blood-brain barrier permeability, and liver toxicity have been validated, and data have been generated for more than 165 chemical compounds to date. Compounds with favorable ADMET profiles can then be transitioned to second tier in vitro testing for cardiotoxicity, advanced liver toxicity, metabolite identification, and protein binding.
Using current approaches, it can take months or years to understand the mechanism of action for new drugs or toxic agents, leaving personnel at risk of exposure to these agents unprotected in the meantime. To address this need, the Defense Advanced Research Projects Agency established the Rapid Threat Assessment program. The program’s goal is to use new high-throughput analytics and mass spectrometry approaches to reduce the time needed to understand a new chemical’s mechanism of action to 30 days. In addition to rapidly providing information on potential toxicity of new chemicals, the approach is expected to reduce the need for animal tests. A proof-of-concept study using this approach successfully identified the mechanism of action of the nitrogen mustard chemotherapeutic agent bendamustine. This five-year partnership with three academic laboratories is scheduled to conclude in late 2018 and will result in publications of methods and techniques.
The Defense Advanced Research Projects Agency’s human-on-a-chip project is developing a microphysiological organ systems platform to evaluate the efficacy and safety of medical countermeasures to toxic agent exposures. This platform includes components representing 10 human physiological systems that interact with each other in a physiologically relevant manner, while maintaining tissue viability for at least four weeks. FDA has been involved in this program from the beginning to ensure that regulatory challenges of drug safety and efficacy review are considered during development. The program will conclude in 2018, and avenues for commercializing the platform are anticipated via Emulate, Inc., and CN Bio Innovations.
The Defense Threat Reduction Agency’s Ex Vivo Countermeasure Evolution and Licensure (XCEL) program develops microphysiological systems to evaluate chemical and biological threat agent assessment and medical countermeasure research and development. XCEL is developing four human primary cell-based organ systems (liver, heart, lung, and kidney/blood vessel) that will be integrated into a platform that includes a blood surrogate, interlinked microfluidics (channels, pumps, and valves), in-line sensors, and off-line analytics with on-board data integration. The lung component of the system, known as the Pulmonary Lung Model, or PuLMo, was recognized by R&D 100 Magazine as a Top 100 Technology Development for 2016. Current efforts are focusing on partial validation of the platform by live testing with known threat agents and toxic drugs.
Occupational and environmental chemical exposures in deployment and training are the most challenging aspects of DOD risk management. Toxicology studies depending on animals are ill-equipped to meet the critical and growing need for data to inform risk management. To address this need, USAF research scientists collaborated with Sanford Burnham Prebys to develop the Cellular Sentinels of Toxicity Platform (CSTP), a suite of HTS assays for cellular phenotype and physiology. CSTP builds upon other high-throughput toxicity screening efforts like ToxCast and Tox21 in two ways. First, CSTP assays utilize human-induced pluripotent stem cells, which more accurately reflect the physiology of mature cells than traditional immortalized cell lines, and thus provide more relevant models for interrogating the effects of potentially toxic agents on the human nervous system, heart, and liver tissue. Second, CSTP combines functional analyses such as cellular impedance and phenotypic readouts that do not require the engineering of genetic constructs to report on cellular function. These assays allow for the early assessment of potential toxicities in a rapid, cost-effective manner that will simultaneously improve the relevance of the data produced while reducing the downstream use of animal studies.
With the goal of unbiased characterization of genetic influence in toxicological exposure risk, USAF scientists developed a high-content assay in which genetically characterized cell lines were treated with a test panel of toxicants. This project, initiated in 2016, is aimed at developing personalized prediction and response of toxicological field exposures, a critical DOD focus area. More than 11,000 morphological features on every cell in the assay were measured, and novel software was developed to identify subsets of these features that can identify exposure to different compounds. During 2016 and 2017, nearly 300 cell lines were screened using high-throughput microscopy, and software was developed to analyze the images, measure and optimize phenotypic features, and identify genetic elements underscoring different toxicological responses. The completion and validation of this assay will enable targeted and sophisticated characterization of toxicant exposure in model cell lines representing any tissue in the human body, thus reducing or eliminating the need for animal studies.
The USAF used high-content in vitro models to obtain data on a large number of chemicals that are relevant to the USAF occupational exposure, either for ground crews maintaining or repairing aircraft or aircrew members during flight. These in vitro cellular systems may inform potential toxicity hazards of hundreds to thousands of chemicals for which there is limited, or no, specific endpoint. Assays using immortal and human stem cells have been used to test thousands of chemicals at many different doses, supporting a mechanistic understanding of toxicity and providing input to quantitative structure-activity relationship (QSAR) and pharmacokinetic models. Recently, advanced algorithms utilizing QSAR and read-across techniques have been applied to Tox21/ToxCast data sets and data from USAF studies using human nervous system stem cells. The goal of these studies is to derive a rapid assessment tool to determine if chemicals measured in the USAF airman breathing space could result in neurological or cognitive effects. Similar approaches have been applied to data sets from a number of chemical toxicological databases to target follow-up studies on only those chemicals having highest toxicity potential.
In April 2017, FDA announced a multiyear research and development agreement with Emulate, Inc., to evaluate the company’s organs-on-chips technology in laboratories at the FDA Center for Food Safety and Applied Nutrition. The project will focus first on developing a liver chip, but the agreement may expand in the future to develop kidney, lung, and intestine models. The ultimate goal is to more precisely predict how specific organs will respond to potential chemical hazards found in foods, cosmetics, or dietary supplements than with current methods. More details about the agreement are available in an FDA blog article by FDA ICCVAM representative Suzanne Fitzpatrick, Ph.D.
Medical device materials contain substances that can produce adverse health effects in patients if released from the device in sufficient quantities. Historically, the potential for health hazards to occur due to the release, or leaching, of these substances from the device was evaluated primarily through animal testing. However, a chemical characterization/risk assessment approach is being increasingly used to assess the potential for adverse systemic effects to occur in patients due to leaching from medical devices. A key component of this approach is exposure assessment; however, data are often unavailable on the amount of the leachables released from medical devices and taken up by the patient. To address this data need, the FDA Center for Devices and Radiological Health (CDRH) is developing computational models to predict patient exposure to compounds released from polymeric materials and have developed a software tool to automatically compare the computer-derived exposure estimate to acceptable exposure limits for the compounds. Based on this comparison, the toxicological risk posed to a patient by any leachable substance can be rapidly assessed, obviating the need for animal testing to assess these biocompatibility endpoints. In 2016, this project focused on developing a computational model to predict the release of color additives from various polymers and to compare the predicted patient exposure to tolerable intake values for the color additives. A beta version of the computational tool was piloted by industry stakeholders in 2017, and the computational model was described in three peer-reviewed journal articles published in 2017 (Chandrasekar et al., 2017a; Chandrasekar et al., 2017b; Janes et al. 2017). Future work will expand the exposure model to include other types of device leachables.
Extractables are substances that can be released from a medical device or material using solvents or conditions that are expected to be at least as aggressive as the conditions of clinical use. These substances can produce adverse health effects in patients if released from the device in sufficient quantities as leachables. A chemical characterization/risk assessment approach is being increasingly used for the biological safety assessment of medical devices, which is expected to reduce use of animals for toxicity and biocompatibility testing. A key component of this approach is the extraction of the device in various solvents, followed by identification and quantification of the extracted compounds. The toxicological safety of the device is then determined by comparing the amount of each compound extracted to an acceptable level of exposure. One limitation of this approach is the lack of clear guidance for conducting the extraction of the device and chemical analysis of the extracts. To address this limitation, in 2017, CDRH began conducting strategic scientific studies that will enable FDA to recommend optimal analytical methods to conduct these chemical analyses. Analytical conditions are being optimized for chemical characterization of a group of six device materials. Optimization includes, but is not limited to, extraction conditions and utilization of up-to-date reference data for identification of extractables and quantification using internal surrogate standards. CDRH organized three seminars within FDA to share initial findings, which will also be presented at the Society of Toxicology meeting in March 2018 (Sussmann et al.). This work will improve public health outcomes while reducing the costs for industry in delivering safe and effective medical devices/technologies to market.
Scientists at NICEATM and the NIH National Center for Advancing Translational Sciences (NCATS) and academic and commercial collaborators used four experimental approaches to better characterize compounds identified in Tox21 quantitative HTS assays as having farnesoid X receptor alpha agonist or antagonist activity. The study generally confirmed the Tox21 results, provided orthogonal data on protein-to-protein interactions and receptor docking, and translated those results to an in vivo system (larval medaka assay). The study, which will be presented at the 2018 Society of Toxicology meeting (Hamm et al.), demonstrates an approach to evaluate compounds that show activity in HTS.
NCATS is leading the Tissue Chip for Drug Screening program in collaboration with other federal government offices to develop human tissue chips. Tissue chips that accurately model the structure and function of human organs will help predict drug safety in humans more rapidly and effectively. The current focus of the program is toxicity testing.
In 2014, NIH and the Defense Advanced Research Projects Agency funded 11 academic laboratories for the second phase of the Tissue Chip for Drug Screening program. The goal of this phase, which concluded in 2017, was to integrate chip devices into a full body system to evaluate drugs and diseases. As part of this program, NCATS established three Tissue Chip Testing Centers in October 2016. The goals for the Tissue Chip Testing Centers include providing the means for scientists participating in the Tissue Chip for Drug Screening program to test and validate tissue chip platforms independently; ensuring wide-ranging availability of tissue chip technology, particularly for regulatory agencies and pharmaceutical companies; and promoting adoption of this technology by the broad research community.
In October 2016, NCATS and the Center for the Advancement of Science in Space announced a funding opportunity to create tissue-on-chip and organ-on-chip platforms that mimic human physiology under the extreme environment of space. Five initial two-year awards were issued in June 2017. The goal of the Tissue Chips in Space initiative is to create tissue-on-chip and organ-on-chip platforms that can be sent to the International Space Station United States National Laboratory (ISS-NL) so that scientists can better understand the role of microgravity on human health and diseases and translate those findings to improve human health on Earth. During the first phase of the initiative, researchers will develop and test tissue chips on the ISS-NL in a microgravity environment. In the second phase, they will further demonstrate the functional use of the tissue chip models for more defined experiments on the ISS-NL.
Cytotoxicity assays are commonly employed as screening tools to identify potential hazards associated with new chemicals and materials. These assays can reduce the need for animal testing in industrial manufacturing. Although cytotoxicity assays are used to evaluate biological effects associated with engineered nanomaterials, several studies that suggest the unique properties of nanomaterials, such as their high surface-to-volume ratio and small size, contribute to significant variability in the assay results. Thus, the need to improve cell-based toxicity assays for nanomaterials is broadly recognized.
To address this problem, NIST scientists used the MTS cell viability assay as a model system. A standard operating procedure for a 96-well assay was developed that included cell line identification testing, dosing preparation, and prescribed pipetting procedures. The protocol incorporates a measurement system that included in-line process controls for reagent quality, cell seeding quality, cell function, and nanomaterial interference. Execution of the protocol produces a value characterizing test nanoparticle toxicity and six additional measurements that characterize the measurement system.
The measurement paradigm was tested in an international study including laboratories from Switzerland, Thailand, the European Union, and Korea. The results indicated several sources of variability in the assay protocol and suggested performance specifications. The study highlighted the critical importance of cell line identification, consistent protocols for rinsing attached cell layers, and detailed nanoparticle dispersion techniques to obtain consistent, reproducible results in these assays. These findings were reflected a final draft international standard, ISO/DIS 19007: Nanotechnologies – In vitro MTS assay for measuring the cytotoxic effect of nanoparticles, which will be published in 2018.
(Photo courtesy of National Institutes of Health)