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Acute systemic toxicity tests are commonly used to evaluate the potential hazards of chemicals, medical products, and other substances via three routes of exposure: ingested (oral toxicity tests), absorbed through the skin (dermal toxicity tests), or inhaled (inhalation toxicity tests). Testing substances to characterize their toxicity provides data that can be used to develop product protective packaging and warning labels, personal protective equipment requirements, and environmental release guidelines.
Acute systemic toxicity tests provide an LD50 or LC50 value, representing respectively the dose (for oral and dermal tests) or concentration (for inhalation tests) that would be expected to produce lethality in 50 percent of the animals tested. The LD50 or LC50 value is used to assign substances to toxicity categories that determine language for product labels to indicate precautions to be taken while handling. (LD50 or LC50 may not be used in the assessment of acute systemic toxicity for some medical products, such as medical devices.)
NICEATM and ICCVAM agencies are working to develop, validate, and implement approaches with the potential to reduce and replace animal use for acute toxicity testing.
High-content screening uses fluorescent tagging and automated imaging to assess changes in the structure and composition of individual cells in a high-throughput manner. The U.S. Army is using high-content screening to screen potential medical countermeasure drugs based on favorable liver toxicity profiles. This two-tiered screening approach uses immortalized HepG2 liver cells and human primary hepatocytes to assess potential drug-induced toxicities. Endpoints assessed in this approach include nuclear changes, mitochondrial inhibition, cell cycle arrest, steatosis, p53 activation, oxidative stress, phospholipidosis, and cell death. Assays used were extensively validated during 2016 and 2017, and now are available for predictive toxicity assessments of potential therapeutics of interest to DOD. Reports of these assessments include graphed results of all measured parameters, a table of curve fit parameters, a human hepatotoxicity risk assessment, and recommendations for follow-up studies. This approach is being expanded to assess cardiotoxicity endpoints such as cardiomyocyte hypertrophy and mitochondrial dysfunction.
USAF needs the ability to rapidly and systematically evaluate toxicity and physiological changes associated with aerospace environments. This task requires versatile in vitro model systems that allow rapid, quantitative, systematic testing while mimicking the in vivo tissue microenvironment. To address this need, the USAF established a multitier evaluation approach that incorporated rapid in vitro screening, mechanistic in vitro studies, limited in vivo studies, and in silico approaches. Human-derived, three-dimensional co-cultures representing the lung, skin, brain, liver, and kidney were developed that include immune cell function. A system specific for respiratory toxicants includes cell models representing the nasal, bronchial, and alveolar regions of the respiratory pathway. Preliminary studies evaluated aerospace toxicology targets, including energetic nanomaterials and heavy metals. Specifically, studies using chromium and cadmium demonstrated region-specific toxicity in the respiratory screen, with soluble metals being more toxic in the nasal region and insoluble metals displaying greater toxicity in the alveolar region. Stem cells are being developed as additional in vitro models to improve predictive capability. The benefits of this approach are lower cost for evaluating physiological and toxicological changes, more rapid screening, use of fewer research animals, and the capability to assess a larger number of experimental conditions with greater predictive power, all of which provide greater protection for personnel working in different operational environments.
In March 2016, EPA published Process for Establishing & Implementing Alternative Approaches to Traditional In Vivo Acute Toxicity Studies for FIFRA Regulatory Use. This guidance document described a transparent, stepwise process for evaluating and implementing alternative methods for the six-pack studies, which test for acute oral, dermal, and inhalation toxicity; skin and eye irritation; and skin sensitization. The document included discussion of three major phases of the evaluation and implementation process, as well as the implications for reporting information required by FIFRA. Establishment of this process and the clear articulation of the related reporting requirements addressed challenges associated with adopting alternative methods.
In November 2016, EPA published Guidance for Waiving Acute Dermal Toxicity Tests for Pesticide Formulations and Supporting Retrospective Analysis. The new guidance expanded the potential for data waivers for acute dermal studies and included a policy statement to waive all acute dermal lethality studies for formulated pesticide products. EPA expects this waiver guidance to reduce the use of laboratory animals, potentially up to 2,500 or more each year.
In December 2016, EPA launched a voluntary pilot program to evaluate the usefulness and acceptability of a mathematical tool that estimates the toxicological classification of a chemical mixture. The mathematical tool, known as the GHS Mixtures Equation, is used in the United Nations Globally Harmonized System of Classification and Labeling of Chemicals (GHS). Use of the GHS Mixtures Equation can reduce animal use for oral and inhalation toxicity studies of pesticide formulations. This program supports the EPA goal of reducing animal testing by adopting better testing methods, as described in the March 2016 Letter to Stakeholders issued by former Office of Pesticide Programs Director Jack Housenger.
EPA requested submission of acute oral and acute inhalation toxicity study data paired with mathematical calculations (GHS Mixtures Equation data) to support the evaluation of pesticide product formulations. The request was accompanied by guidance on how pesticide companies can submit data for the program. At the end of 2017, EPA was still accepting data submissions, with plans to conduct an analysis in 2018.
NICEATM and EPA scientists examined a data set of over 21,000 in vivo LD50 values obtained from multiple curated databases to assess data variability in animal studies. Alternative models developed for estimating acute systemic toxicity are generally evaluated using LD50 values from animal studies that can produce variable results, even when conducted according to accepted test guidelines. Such variability can make assessment of alternative models extremely challenging when these data are used as reference values. Within the LD50 data set examined by NICEATM and EPA, a degree of variability was observed that had the potential to confound hazard categorization. Specifically, LD50 values from 59 chemicals fell into at least three different GHS oral acute toxicity labeling categories, and values from 49 chemicals fell into at least three EPA hazard categories. These findings underscore the importance of considering an appropriate margin of uncertainty when using in vivo oral acute toxicity data to assess the performance of alternative methods.
This data set will be made available in 2018 through the NICEATM Integrated Chemical Environment and provide a reference data set to ensure that appropriately representative LD50 data are routinely used for the development and validation of alternative models. The analysis of the full data set will be presented at the 2018 annual meeting of the Society of Toxicology (Karmaus et al.), and a journal article describing the analysis is in preparation.
Although cytotoxicity assay data cannot currently be used to replace animal tests for predicting acute hazard classes, two in vitro cytotoxicity assays have been validated to estimate starting doses for acute oral toxicity tests in animals. To more broadly investigate the utility of cytotoxicity and other data from in vitro assays to predict acute lethality, NICEATM used HTS data from the ToxCast and Tox21 programs to predict LD50 values and binary toxicity categories (toxic vs. nontoxic). To further investigate the correlation of in vitro to in vivo results, reverse toxicokinetics was used to estimate equivalent administered doses from in vitro effective concentrations. These analyses confirmed that no single in vitro assay can currently predict acute systemic toxicity in rodents. The analyses were presented (Strickland et al.) at the 2017 World Congress on Alternatives and Animal Use in the Life Sciences.
To follow up on these results, NICEATM conducted an analysis using the random forest machine learning method and data from ToxCast and Tox21 to determine whether a specific group of high-throughput assays could be identified that would be informative in predicting oral LD50 values. Preliminary results using a data set including about 1,700 chemicals and about 200 ToxCast and Tox21 assays suggested that the approach might be able to distinguish toxic from nontoxic chemicals, especially if the nontoxic chemicals were conservatively defined as those having LD50 values greater than 5,000 milligrams per kilogram of body weight. Ongoing work will apply this approach to a larger data set, explore the use of other machine learning methods, and revise the approach to consider cheminformatics and mechanistic information.