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Reducing or eliminating animal testing is a key goal of the Strategic Roadmap. To this end, ICCVAM agencies that conduct chemical safety testing develop and use approaches that reduce or replace animal use. Likewise, ICCVAM agencies that require their stakeholders to conduct testing are reducing required animal use by developing criteria for waiving tests.
DOT proposed an amendment to the Hazardous Materials Regulation in November 2018 (83 FR 60970). The amendment would enable non-testing alternatives that use existing data on chemical properties to be considered for classifying corrosive mixtures. Currently, the regulation requires offerors to classify Class 8 corrosive material and assign a packing group based on test data. The regulation authorizes a skin corrosion test and various in vitro test methods that do not involve animal testing. However, only data obtained from testing are currently acceptable for classification and assignment to a packing group. These alternatives would afford offerors the ability to make a classification and packing group assignment without the need to conduct physical tests.
Fisheries managers use toxicants to control invasive and undesirable fish species. The U.S. Geological Survey (USGS) Upper Midwest Environmental Science Center of DOI developed a screening process to minimize and replace animal testing while developing new chemical control agents for invasive species. The goal is to identify compounds that are potentially toxic to target species while posing minimal risk to native species. The three integrated phases of the screening process include (1) identifying physical and chemical properties of compounds that affect bioavailability in fish, (2) prescreening of a publicly available chemical databank to prioritize candidate compounds, and (3) screening of selected compounds for cytotoxicity using in vitro biological assays, ecotoxicity modeling, and fish cell lines. Although in vivo testing continues to be utilized in the development of new fish toxicants, the screening process enables minimization of animal testing while developing a new chemical control agent for invasive species.
Ecotoxicity modeling uses a QSAR modeling system that determines species-specific responses to chemical exposures using existing toxicity data and chemical properties. These in silico assessment methods can be used to prioritize candidate compounds and estimate cytotoxicity. Models can predict more than 3,000 endpoints for over 600 taxa against more than 9,000 chemicals. A user interactive prototype of the model results is available on the USGS website.
Promising novel toxicants identified in the ecotoxicity modeling step are tested using cellular assays to identify which toxicants have the biological activity required. USGS has developed multiple endpoint assays that measure cell viability based on quantitation of adenosine triphosphate. These assays use cell lines from native species including fathead minnow, bluegill sunfish, rainbow trout, lake sturgeon, and paddlefish, as well as invasive species, including silver carp and bighead carp. Six novel species-selective toxicants have been identified. Four toxicants demonstrating potent species-selective cytotoxic effects are being manufactured for in vivo toxicity screening assays. Two toxicants have completed in vivo testing and show modest selectivity for silver and bighead carp.
As part of ongoing assessments of wildlife health, DOI is investigating potential cardiovascular effects on fish from pesticides and pharmaceuticals frequently detected in surface waters and fish tissues. The U.S. Geological Survey's Columbia Environmental Research Center conducts high-content screening of compounds to formulate hypotheses and prioritize compounds for further toxicity testing. This approach reduces animal use, test compound needed, and waste by utilizing pre-feeding fish embryos in a microtiter plate format. This approach is also being used to better characterize toxicity of PAHs and oxygenated PAHs in groundwater samples over different trophic levels of a subsurface oil spill. These assays can provide evidence to justify larger-scale studies to determine actual risk versus perceived risk of contaminants.
The Center’s current high-content screening assay is a developmental cardiotoxicity assay that assesses total body length, pericardial area, intersegmental vessel area, circulation, and heart rate after a 72-hour exposure. This array of endpoints allows for a targeted assessment of toxicity. In addition to an LC50 estimate, the assay rapidly provides mode-of-action information, allowing formation of hypotheses on sublethal impacts of contaminants. Data derived from these studies on acute toxicity and mode of action for pesticides, pharmaceuticals, and PAHs/oxygenated PAHs will support a better understanding of potential effects on wildlife species.
DOI utilizes cell-based bioassays for HTS of contaminants in environmental samples. The cell-based bioassays at the U.S. Geological Survey's Columbia Environmental Research Center are responsive to dioxin-like chemicals, PAHs, and a variety of endocrine-active substances such as estrogenic, androgenic, glucocorticoid-like, progestogenic, and steroidogenic chemicals. HTS is a valuable tool for quickly and inexpensively identifying bioactive samples for further study in higher-tier assays. DOI utilizes these assays for mapping PAH-like activity in oil-contaminated groundwater, mapping estrogenic activity in sediments from the Upper Coosa River Basin, quantitating estrone in laboratory water samples, assessing fish feed for estrogenic activity, testing pure chemicals and chemical mixtures for bioactivity, and guiding chemical analysis of endocrine-active chemicals in an effects-directed analysis of sediments and water from the Chesapeake Bay watershed. Currently, nontargeted analysis of bioactive fractions from the Chesapeake Bay watershed sediments is underway for identification of endocrine-active chemicals. A similar approach will be used to identify oxygenated PAHs from oil-contaminated groundwater. Several of the cell bioassays are also undergoing mechanistic evaluation for expanded utility in screening additional types of contaminants. The cell bioassays at the Center are primarily luciferase transactivation cell bioassays and include cell lines from the EPA’s Endocrine Disruptor Screening Program and Tox21.
Cyanobacteria, also known as blue-green algae, produce potent toxins called microcystins. During algal blooms, concentrations of microcystins can reach levels that are toxic to vertebrates and adversely affect fish and wildlife health. While acute effects of high-dose microcystin exposure have been investigated, there has been less focus on adverse effects resulting from low-dose or chronic exposure. In addition, there are few options for testing effects on non-model organisms. During 2018-2019, scientists in the U.S. Geological Survey of the DOI developed and applied methods to evaluate cellular and molecular responses to microcystins using primary tissue culture approaches. These in vitro methods include evaluation of primary hepatocytes and leukocytes from fishes that inhabit aquatic ecosystems vulnerable to harmful algal blooms. Specifically, this research focused on smallmouth bass collected from the upper Chesapeake Bay watershed. This analytical approach includes the application of in vitro exposures, image analysis-based flow cytometry, and transcriptional profiling of hepatic and immune-responsive genes using nCounter technology. These in vitro approaches, used to interrogate specific mechanistic questions in environmentally relevant fishes, minimize the use of vertebrates.
Chemical contaminants are introduced to environmental waters via many sources, and many of these contaminants have the potential to adversely affect organisms living in these waters. Recognized adverse effects include the induction of cancer via genotoxic mechanisms, endocrine disruption via the derailment of normal hormone signaling pathways, and outright toxicity leading to disease or death. Scientists in the U.S. Geological Survey (USGS) within DOI have established or adapted water collection, extraction, and in vitro screening assays to evaluate the bioactivity of surface water samples. These approaches circumvent the need to utilize vertebrates and minimize endpoint variability in bioactivity measures. Data from these assays are incorporated into predictive modeling analyses to identify land uses associated with predicted biological disruption. In addition, they are also applied to responsibly inform site selection for comprehensive environmental sampling. During 2018-2019 these assays were applied to augment USGS and other collaborator data sets collected from environmental surface and well waters collected in the eastern and midwestern United States.
In a September 2019 news release, EPA announced a directive to prioritize efforts to reduce animal testing. The directive, issued by EPA Administrator Andrew Wheeler, called for reducing mammal study requests and funding 30% by 2025 and completely eliminating them by 2035. Wheeler also announced $4.25 million in funding to five universities to research the development and use of alternative test methods and strategies that reduce, refine, and/or replace vertebrate animal testing. Studies funded include in vitro models to test for developmental and reproductive toxicants, neurotoxins, and toxicity of complex environmental mixtures.
In a September 2019 news release, EPA announced a draft science policy to reduce testing of pesticides on birds when registering conventional outdoor pesticides. The draft policy describes the results and implications of a retrospective study conducted by EPA and People for the Ethical Treatment of Animals. The study explored the quantitative and qualitative contributions of risk assessment methods using single oral dose and subacute dietary toxicity endpoints to the overall conclusions of acute avian risk. The analysis indicated that, in most cases, the subacute dietary results had little impact on risk conclusions based on the use of acute oral data alone. This finding is expected to reduce the number of animals tested by a total of 60 birds per test, for a total projected animal savings of over 700 animals per year.
An article in Regulatory Toxicology and Pharmacology co-authored by ICCVAM co-chair Anna Lowit (Craig et al. 2019) summarizes the activities of EPA's Hazard and Science Policy Council (HASPOC). HASPOC was established in 2012 by the EPA Office of Pesticide Programs to consider requests for waiving animal study requirements for human health risk assessments. Since its inception, HASPOC has evaluated over 1,000 requests to waive animal studies and granted waivers in response to nearly 90% of requests. As of the article’s publication in 2019, these waivers have saved over 200,000 animals, $300 million in study costs, and $6 million in study review costs.
As FDA advances its Predictive Toxicology Roadmap, its Center for Food Safety and Applied Nutrition will reevaluate traditional approaches to toxicity testing to refine testing recommendations and include accurate predictive models of toxicity that are appropriate to use in evaluating the safe use of food additives or color additives. As part of this effort, the Center reviewed all dog studies previously submitted as part of the approval for food additive or color additive petitions. These findings may enable the Center to determine when a toxicity study in dogs could provide toxicological information not otherwise available using other experimental model systems and may help to identify endpoints for potential evaluation using modern in vitro or in silico techniques. A report on the findings of the review is in preparation.
USDA is committed to reducing the use of live animals in testing and experimentation. Vaccines used in controlling animal disease frequently undergo testing in animal models to ensure they are effective. The USDA Center for Veterinary Biologics (CVB) enforces the Virus Serum Toxin Act, which requires animal vaccines to be safe, potent, and effective. From 2013-2018, CVB developed alternatives to the codified Leptospira vaccine potency test to help reduce the number of hamsters used for this test. Hamsters are used because of their sensitivity to the Leptospira organism. Approximately 40 total hamsters per serogroup are required for potency testing each leptospiral fraction. This number encompasses 10 vaccinated animals, 10 unvaccinated control animals, and additional unvaccinated animals used to determine the LD50 of the challenge strain. A valid test that confirms sufficient vaccine potency requires 80% survival in the vaccinated animals and 80% mortality in unvaccinated control animals after administering a standardized dose of leptospiral challenge.
In order to reduce the number of animals used in this testing, CVB provided options to veterinary biologics manufacturers. An ELISA was developed by CVB to eliminate live animal potency testing after appropriate validation for a particular product line. In cases where the ELISA was not yet a reasonable option for a product line, CVB allowed back-titration hamsters to be removed from the codified test. A company that wishes to use these options must request and receive an exemption from CVB.
A separate group of hamsters is also required to propagate and maintain virulent strains for the codified test and developmental needs associated with regulated vaccines. Over 2,500 hamsters per year per facility are estimated to be used for propagation of virulent Leptospira. As a result, the CVB developed a cryopreservation protocol for the commonly used leptospiral strains and provides cryopreserved virulent Leptospira upon request.
From 2013 to 2018, the USDA Animal Care program monitored the number of hamsters listed under Category E on the annual reports from six companies that conduct Leptospira vaccine potency testing. Category E includes instances in which pain or distress, or potential pain or distress, is not relieved with anesthetics, analgesics, and/or tranquilizer drugs. Facilities that use animals for research, teaching, and testing are required to submit to the USDA an annual report on animal usage under the Animal Welfare Act. In 2013, the Category E designations indicated approximately 35,767 hamsters were used in total for Leptospira vaccine potency testing. Monitoring revealed a steady decline in animal numbers over five years such that 20,099 hamsters were used in 2018 demonstrating a 38% reduction.
CVB believes the findings indicate that these options significantly contributed to the downward trend in hamster use. The options remain available and can be found on the CVB website. At this juncture, the USDA will continue annual monitoring of hamster use in Leptospira vaccine potency testing and also explore other areas where the 3Rs (replacement, reduction, or refinement of animal use) can be applied.
For the past 40 years, questions have been raised about the relevance and regulatory utility of rodent cancer bioassays in human health risk assessment. To address these questions, EPA collaborated with experts from industry and animal welfare stakeholder groups to form the Rethinking Carcinogenicity Assessment for Agrochemicals Project. The project’s goal is to determine both the appropriateness of and criteria for waiving rodent cancer bioassays for the registration of food-use pesticides. A weight-of-evidence reporting framework is being developed to determine when sufficient information is available to perform a health-protective chronic risk assessment without conducting rodent cancer bioassays. Information used includes exposure, mode-of-action, physiochemical properties, metabolism, and subchronic toxicological data from standard risk assessment endpoints. An abstract describing the framework criteria and example carcinogenicity waivers (Hilton et al.) was accepted for presentation at the 2020 Society of Toxicology meeting.