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ICCVAM Biennial Report 2018-2019

Biennial Progress Report 2018-2019 Interagency Coordinating Committee on the Validation of Alternative Methods

Assay Application

ICCVAM and its member agencies conduct, support, and evaluate studies investigating the suitability of new laboratory methods for specific purposes. Methods evaluated during 2018 and 2019 addressed endpoints including inhalation toxicity, skin sensitization, eye irritation, and endocrine disruption.

Electrophilic Allergen Screening Assay Validation Study

Binding of a chemical to skin proteins is the first step in the development of allergic contact dermatitis. The electrophilic allergen screening assay is a chemical assay that measures light absorbance or a fluorescent signal in proportion to a chemical’s tendency to bind to proteins.

The electrophilic allergen screening assay, developed by scientists at NIOSH, was nominated to ICCVAM to evaluate its usefulness for identifying potential skin sensitizers. Four ICCVAM member agencies are participating in an ongoing validation study of the assay. Testing of 10 chemicals during 2018 showed that the method has sufficiently good reproducibility and accuracy rates to support further evaluation. In 2019, CPSC and NIST modified the assay to a 96-well format to increase throughput and accessibility of the assay; details of this process are described in an abstract (Gordon et al.) accepted for presentation at the Society of Toxicology 2020 annual meeting. Testing of 20 chemicals will be conducted using the 96-well assay during 2020.

EpiAirway Validation Study

Acute inhalation systemic toxicity tests identify substances that could cause illness or death after a single inhaled exposure. NICEATM facilitates interactions among stakeholder groups interested in developing and promoting alternatives to animal use for these tests.

A cooperative agreement under the NIEHS Phase IIb Small Business Innovation Research program provided funding to MatTek Corporation to validate its EpiAirway™ in vitro human bronchial tissue model to predict the toxicity of inhaled chemicals. Testing of reference chemicals to determine the usefulness and limitations of EpiAirway for this purpose has been completed. Several ICCVAM agency representatives are members of the cooperative agreement steering committee.

OptiSafe Validation Study

NICEATM coordinated a multi-laboratory validation study to determine the reliability and relevance of the OptiSafe test method. In this method, a test substance is applied to a semi-permeable membrane to assess the substance’s potential to cause eye irritation.

The study was completed in 2018 and a report on the study has been accepted for publication (Choksi et al. 2020). The study demonstrated that the OptiSafe method is useful for identifying non-surfactant substances that do not require classification for ocular irritancy and thus can reduce the use of animals for this type of testing.

Review and Comment on the Genomic Allergen Rapid Detection Assay

The ICCVAM Skin Sensitization Workgroup reviewed the validation study report of the Genomic Allergen Rapid Detection™ assay for skin sensitization potential. This method measures changes in gene expression for 200 genes relevant to skin sensitization in a human myeloid leukemia cell line after the exposure of the cells to test substances.

The review of the validation study report started in February 2018. The ICCVAM workgroup did not support a further comprehensive assessment and peer review of this method, based on a number of considerations:

  • Raw data from the study and subsequent nanoString analyses were not provided. A peer review cannot be conducted without access to raw data, necessary to evaluate the appropriateness and consistency of data analysis and processing.
  • The prediction algorithm was not disclosed. This precluded a review of model features typically evaluated by peer review (e.g., statistical robustness of methods used).
  • Eleven of the 28 chemicals used to develop the prediction model were used to evaluate the performance of the model. Chemicals used to develop the prediction model should not be used in an independent evaluation of model accuracy.
  • The applicability domain of the method was not defined.

ICCVAM provided a response to test method developers in April 2018.

Evaluation of a Predictive Model of Developmental Vascular Toxicity

NICEATM and EPA scientists built an AOP for quantitative prediction of developmental vascular toxicity. This AOP was then applied to ToxCast HTS data to develop predictions of chemicals’ potential to disrupt angiogenesis, or blood vessel development (Saili et al. 2019). The predictions were evaluated for 38 compounds tested across a suite of functional assays for the angiogenic cycle, including assays in complex cell systems, virtual tissues, and small model organisms. The results serve to boost confidence in the capacity of HTS data to predict developmental vascular toxicity.

In Vitro Testing to Support Development of Defined Approaches for Eye Irritation of Agricultural Products

NICEATM, the PETA International Science Consortium Ltd., EPA, and CropLife America member companies are collaborating to develop an in vitro defined approach for hazard classification of eye irritation potential of agrochemical formulations. A three-phased prospective evaluation was designed to (1) assess the applicability of seven in vitro eye irritation/corrosion protocols to agrochemical formulations and (2) develop a defined approach for agrochemical formulations testing for prediction of U.S. and international irritancy classifications. In Phase 1, completed in 2018, six formulations were tested in seven different eye irritation test protocols. Ten additional agrochemical formulations with in vivo data representing a wider range of eye irritation classifications were evaluated in Phase 2 during 2019. While none of the methods directly correlated with the in vivo results, several methods showed potential for use in a defined approach. Analysis of the data from Phases 1 and 2 will determine the next steps of the study. An abstract describing this work (Choksi et al.) was accepted for presentation at the 2020 Society of Toxicology annual meeting.

Validation of Androgen Receptor Activity Assays

NICEATM collaborated with test method developer CertiChem, Inc. to validate an in vitro test method that uses MDA-Kb2 human breast cancer cells to measure androgen receptor agonist and antagonist activity. Specifically, NICEATM provided guidance on incorporating a cytotoxicity assay into the test method protocol. Testing of 67 coded reference chemicals in agonist and antagonist modes to characterize method reliability and relevance is finished, and a report summarizing these results was provided to CertiChem in December of 2018.

Collaboration with Cosmetics Europe to Evaluate Defined Approaches for Skin Sensitization

NICEATM and Cosmetics Europe collaborated to evaluate multiple defined approaches for skin sensitization safety assessment that had been submitted to OECD. The collaboration produced two publications:

  • Hoffman et al. 2018 describes a database including data from human, animal, and five non-animal tests for 128 substances. The substances in the database have a variety of chemical structures and use categories. The database is a proposed point of reference for the evaluation and development of new non-animal approaches to skin sensitization safety assessment.
  • Kleinstreuer et al. 2018 describes an analysis of multiple defined approaches for skin sensitization safety assessment of cosmetic ingredients using the database described in Hoffman et al. Many of these approaches were found to perform as well or better than animal methods to predict human skin sensitization hazards.

Based on the results of these analyses, NTP has adopted in vitro skin sensitization methods and associated defined approaches as the default approach to assess the potential for chemicals to cause allergic contact dermatitis.

Interlaboratory Study to Examine Effects of Key Protocol Elements for Zebrafish Developmental Toxicity Studies

To enable broader adoption of zebrafish for toxicological screening, NTP established the Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) program.

An initial information-gathering phase of the SEAZIT program identified a need for an interlaboratory study to more closely examine the effects of variation in key protocol elements in developmental toxicity studies. This study began in 2019, and participating laboratories are conducting dose range-finding experiments. The study is designed to determine the effect of chorion removal and exposure media renewal on study outcomes. Participating laboratories will use in-house protocols to test a defined chemical set while varying the two protocol elements under investigation. The chemical set, which was designed to provide overlap with other NTP studies, includes chemicals with a range of physicochemical properties and developmental effects. Many of the chemicals have in vivo reference data available from rodent and other zebrafish studies. The interlaboratory study includes a pilot effort on chemical kinetics in support of future studies of absorption, distribution, metabolism, and excretion in zebrafish.

A primary goal of SEAZIT is to develop best practices for data analysis. To this end, the data generated in this study will be made publicly available, so that all study data may be used by investigators to estimate consensus toxicity values for each chemical.

Optimization and Validation of an In Vitro Botulinum Neurotoxin Assay

Tests to detect and measure botulinum neurotoxin are required by multiple federal agencies for a variety of purposes, such as measuring toxin in drug formulations or detecting toxin in possibly contaminated food or wildlife. Currently, the standard test for these endpoints is a mouse lethality assay that can use large numbers of animals. NICEATM supports efforts to develop, validate, and implement alternative approaches for tests used to detect the presence of botulinum neurotoxin and measure potency of botulinum neurotoxin preparations.

NICEATM supported the optimization and validation of ELISAs that replace animal-based methods for diagnosing suspected avian botulism samples. Methods were developed for determining the presence or absence of botulinum neurotoxin serotypes C, D, and E in field-collected samples from a wide range of bird species. These methods may be used in the future to support testing requirements for the U.S. Geological Survey’s National Wildlife Health Center.

Testing to Expand the Applicability Domain of Three In Vitro Skin Sensitization Assays

Defined approaches developed by NICEATM and ICCVAM use non-animal data to predict skin sensitization hazard and potency. These approaches combine data from the in vitro direct peptide reactivity assay (DPRA), KeratinoSens assay, and human cell line activation test (h-CLAT); read-across predictions generated by the QSAR Toolbox software package; and physical properties such as relative solubility in water and organic solvents. The defined approaches were described in papers published in 2016 and 2017 (Strickland et al. 2016, Strickland et al. 2017, Zang et al. 2017).

To assess and expand the potential applicability of these defined approaches to a broader range of chemical types, ICCVAM agencies nominated over 200 chemicals for additional testing in the DPRA, KeratinoSens, and h-CLAT tests. Chemicals being tested include pesticide ingredients and formulations, industrial chemicals, and personal care product ingredients. NTP is conducting this testing, which is expected to be completed in 2020. The study data will enable NICEATM and ICCVAM to evaluate the appropriateness of defined approaches using these three in vitro methods for various regulatory applications.

Addressing Emerging Contaminants

The term NAMs refers to any non-animal technology, methodology, approach, or combination of these that can be used to provide information on chemical hazard and risk assessment. NAMs are being applied to address public concern about exposure and environmental and health effects of emerging contaminants, driven in part by reports of potential human exposures to substances with limited information available on toxicity and exposure. Per- and polyfluoroalkyl substances (PFAS) are examples of such emerging contaminants. These substances present a complex problem, involving multiple chemicals, multiple routes of exposure, and multiple potential human health and ecological outcomes of concern. The hundreds of untested PFAS provide a scenario in which traditional one-by-one toxicity testing would require commitment of tremendous resources and assessment-relevant information would not be available for years. EPA and NTP are generating data through in vitro high-throughput toxicity testing and high-throughput toxicokinetic assays to inform hazard effects characterization and promote prioritization of chemicals for further in vivo testing (Patlewicz et al. 2019). This effort also will address PFAS lacking toxicity information by facilitating read-across approaches to infer the toxicological properties across the broader range of PFAS.

Evaluation of FXR-active Chemicals Identified from Tox21 Screening

Farnesoid X receptor alpha (FXRα) is a member of the nuclear receptor superfamily involved in bile acid homeostasis, glucose metabolism, lipid homeostasis, and hepatic regeneration. NIEHS and NIH scientists with academic and industry collaborators evaluated substances identified in Tox21 HTS in vitro screens as FXRα agonists and antagonists using four experimental approaches. The study generally confirmed quantitative HTS in vitro results, provided data on protein:protein interactions and receptor docking, and translated those results to an in vivo system. A poster describing the study (Hamm et al.) was presented at the 2018 Society of Toxicology annual meeting.

Airman-on-a chip System Applied to Understanding Hyperoxic Oscillations

AFRL is using iPSC technology to develop personalized brain-on-a-chip microfluidic platforms harnessing cells from individual airmen. These platforms will provide insight into how an airman’s genetic background affects resiliency or susceptibility to operational conditions. The brain-on-a-chip platforms are comprised of iPSC-derived gluta- and gaba-minergic neurons, astrocytes, pericytes, and brain microvascular endothelial cells, and can allow investigations of complex blood-brain barrier neurovascular interactions. In current studies, the platforms are exposed to dynamic oxygen conditions simulating a fighter pilot’s oxygen exposure profiles, followed by comprehensive molecular analyses. In parallel, a matched human cohort is being evaluated in a hyperoxic chamber and physiologically and cognitively assessed. This study aligns in vitro work with in vivo human results to predict molecular outcomes and will provide a basis to predict an airman’s resiliency and target enhancement strategies for optimal performance.

Caenorhabditis elegans Assays for Developmental Neurotoxicity

Caenorhabditis elegans are small, non-pathogenic roundworms with many specialized tissues that function in ways that correspond to vertebrate organs. Many cellular and genetic pathways involved in development, neuronal architecture and function, and toxic mode of action are conserved between worms and humans (Hunt 2017). C. elegans’ 3-day lifecycle and ease of maintenance suggest that the organism could be a good candidate model for fast and inexpensive alternatives to mammalian testing, but only if specific assays can be demonstrated to provide data that corresponds to mammalian toxic response. The worm development and activity test, developed by the FDA Center for Food Safety and Applied Nutrition, shows promise for identifying mammalian developmental neurotoxins (Hunt et al. 2018). The test is currently being assessed using a panel of 20 blinded compounds with known developmental and neurotoxicity effects in mammals.

Evaluation of a Proposed Approach to Refine Inhalation Risk Assessment for Point of Contact Toxicity

EPA conducts human health risk assessments to evaluate the potential health effects of pesticides and toxic chemicals based on the use pattern or conditions of use. For evaluating effects via the inhalation route, data are required from subchronic inhalation toxicity studies using animals, usually rats. However, human and rat respiratory tracts differ to an extent that may affect the ability of animal test results to correctly predict effects in humans. It is also challenging to accurately establish a no-observed-adverse-effect concentration from animal studies. To that end, Syngenta Crop Protection proposed using a 3D in vitro inhalation toxicity model to derive a point of departure for inhalation toxicity for the fungicide chlorothalonil. The EPA Office of Pesticide Programs and Office of Pollution Prevention and Toxics provided feedback to Syngenta during development of the proposed approach. Results of the case study were evaluated by the Scientific Advisory Panel for the Federal Insecticide, Fungicide, and Rodenticide Act at their December 2018 meeting. The panel’s report, issued in April 2019, expresses support for the approach and makes recommendations for improvements. EPA has continued to work with Syngenta to address the panel’s recommendations.

Using ToxCast Data for Food Chemical Safety Risk Assessment

NAMs are currently being developed and evaluated for use in chemical safety risk assessment, including chemicals used in food. NAMs include in vitro HTS assays such as those used in ToxCast and Tox21. These assays have been run for thousands of compounds, including hundreds of compounds used in food. However, the relationship of these NAM data with traditional in vivo animal data and the utility of NAMs for risk assessment remain under evaluation. This study evaluated the utility of ToxCast/Tox21 HTS data in food safety risk assessment. To do this, bioactive concentrations of a subset of food-use compounds in ToxCast were converted to oral equivalent doses via IVIVE using either in vitro or in silico-based toxicokinetic parameters for a subset of food-use compounds. These oral equivalent doses were then compared to doses demonstrated to cause effects in in vivo animal tests using data compiled by EPA and FDA. Initial comparisons demonstrated great variability in the correlation between ToxCast and in vivo data, so steps are being taken to further refine the toxicokinetic information, chemical groups, and in vivo endpoints to identify additional information and conditions necessary to utilize HTS data for preliminary food safety assessment.