The In Vitro and Alternative Methods Specialty Section (IVAM) is pleased to present recordings of its series of webinars focused upon the development and application of in vitro methods and non-animal models for toxicology and product safety.
Date and Time: Wednesday, May 19, 2021 at 11:00 AM ET
Duration: 1 hour
Presenter: Vinicius Alves, PhD, Researcher Assistant Professor, Division of Chemical Biology and Medicinal Chemistry, University of North Carolina at Chapel Hill
Title: Pred-Skin: A Web Portal for Accurate Prediction of Human Skin Sensitizers
Safety assessment is an essential component of the regulatory acceptance of industrial chemicals. Previously, we have developed a model to predict the skin sensitization potential of chemicals for two assays, the human patch test and murine local lymph node assay and implemented this model in a web portal. Here, we report on the substantially revised and expanded freely available web tool, Pred-Skin version 3.0. This up-to-date version of Pred-Skin incorporates multiple quantitative structure–activity relationship (QSAR) models developed with in vitro, in chemico, and mice and human in vivo data, integrated into a consensus naïve Bayes model that predicts human effects. Individual QSAR models were generated using skin sensitization data derived from human repeat insult patch tests, human maximization tests, and mouse local lymph node assays. In addition, data for three validated alternative methods, the direct peptide reactivity assay, KeratinoSens, and the human cell line activation test, were employed as well. Models were developed using open-source tools and rigorously validated according to the best practices of QSAR modeling. Predictions obtained from these models were then used to build a naïve Bayes model for predicting human skin sensitization with the following external prediction accuracy: correct classification rate (89%), sensitivity (94%), positive predicted value (91%), specificity (84%), and negative predicted value (89%). As an additional assessment of model performance, we identified 11 cosmetic ingredients known to cause skin sensitization but were not included in our training set, and nine of them were accurately predicted as sensitizers by our models. Pred-Skin can be used as a reliable alternative to animal tests for predicting human skin sensitization.
Presenter: Emily Golden, MS Graduate Student, Center for Alternatives to Animal Testing, Johns Hopkins University
Title: Evaluation of the Global Performance of Eight In Silico Skin Sensitization Models Using Human Data
Allergic contact dermatitis, or the clinical manifestation of skin sensitization, is a leading occupational hazard. Several testing approaches exist to assess skin sensitization, but in silico models are perhaps the most advantageous due to their high speed and low-cost results. Many in silico skin sensitization models exist, though many have only been tested against results from animal studies (e.g., LLNA); this creates uncertainty in human skin sensitization assessments in both a screening and regulatory context. This project’s aim was to evaluate the accuracy of eight in silico skin sensitization models against two human data sets: one highly curated (Basketter et al., 2014) and one screening level (HSDB). The binary skin sensitization status of each chemical in each of’s QSAR Toolbox, UL’s REACHAcross™, Danish QSAR Database, TIMES-SS, and Lhasa Limited’s Derek Nexus). Models were assessed for coverage, accuracy, sensitivity, and specificity, as well as optimization features (e.g., probability of accuracy, applicability domain, etc.), if available. While there was a wide range of sensitivity and specificity, the models generally performed comparably to the LLNA in predicting human skin sensitization status (i.e., approximately 70–80% accuracy). Additionally, the models did not mispredict the same compounds, suggesting there might be an advantage in combining models. In silico skin sensitization models offer accurate and useful insights in a screening context; however, further improvements are necessary so these models may be considered fully reliable for regulatory applications.
Presenter: Glenn Myatt, PhD, CEO, Leadscope, Inc.
Title: Skin Sensitization In Silico Protocol
In silico toxicology protocol for skin sensitization computational frameworks that incorporate in silico best practices will have a critical role in expanding the application of computational toxicology. The in silico toxicology protocol consortium is one example which is developing a series of protocols (equivalent to experimental test guidelines) to support the acceptance and adoption of such methods. A general framework for organizing such protocols has been published with endpoint-specific protocols completed for genetic toxicology and skin sensitization. A series of additional protocols are in development. The skin sensitization protocol incorporates recent advances in the understanding of key events along the adverse outcome pathway. These events, along with other supporting information, are incorporated into the in silico toxicology hazard assessment framework. Information from both experimental data and/or in silico model results for individual effects or mechanisms are incorporated within this framework. Based on the relevance of the mechanisms and effects as well as the strengths and limitations of the experimental systems used to identify them, rules and principles are defined for deriving skin sensitization assessments based on the weight of the information. Further, the assignments of reliability and confidence scores that reflect the overall strength of the assessment are discussed. This presentation will provide an overview of the in silico toxicology protocol framework, describe the skin sensitization hazard assessment framework and present a worked example.
Registration is required.
Date: Friday, January 29, 2021
Thomas Hartung, MD, PhD, Center for Alternatives to Animal Testing, Johns Hopkins University
Title: A.I. and Big Data—New Kids on the Toxicology Block
Abstract: Big data and machine learning (A.I.) have impacted on every aspect of our life in recent years. Simply said, A.I. is making big sense from big data. Not different, toxicology sees the rise of big data, e.g. the use of ~omics technologies, high-content imaging, sensor technologies, robotized testing such as by ToxCast and the Tox21 alliance, curated legacy databases, scientific publications, the grey literature of the internet etc., which all feed these big data. The magic of A.I. is that more and more of this can be brought together to integrate these pieces of information, which is called data fusion or transfer learning. In 2018, we pushed this even further by introducing automated read-across into this modelling, i.e. taking advantage of the fact that chemical similarity is bringing additional structures into the data. At the basis, a map of the chemical universe was created based on 10 million structures, where similar chemicals are close and dissimilar ones are far from each other, the distance reflecting their degree of similarity. The resulting map allows us to place any chemical in its similarity space and not only interpolate from its neighbors over all their properties but also to assign a certainty of the prediction based on the specific constellation of information. In simple terms, if there is non-contradictory information from many closely similar structures, the result is pretty certain. The resulting read-across-based structure activity relationship (RASAR) already in its first implementation impressed with a balanced accuracy of 87% for 190,000 cases of chemicals with known hazard classifications. This compares favorably with only 81% reproducibility of six OECD animal guideline tests in a subset of the database. This approach, based on only curated legacy databases, has been expanded by us and others since 2018. The vision is to integrate more types of information and increasingly use biological similarity, not only the similarity of chemical structures with respect to shared functional groups. This wholesome evidence integration brings a tool to the hands of the practitioner of toxicology to support the analysis of untested or large groups of substances. Formal evaluations are on the way and with Australia’s new chemical legislation, the first regulatory acceptance has just been achieved.
Thomas Luechtefeld, PhD, Insilica LLC
Title: Smart Chemistry—Algorithms for Chemical Classification and Clustering
Abstract: Chemical data is growing quickly, with thousands of new tests and millions of chemicals tested each year. However, most chemical property models focus on results from a handful of test types and fail to leverage the vast majority of available data. Neural networks enable transfer learning where models trained on one task accelerate learning on other tasks and the rapid expansion of chemical data increases the value of transfer learning between chemical property domains. Models of eye irritation can benefit from models of skin irritation via transfer learning. This kind of transfer learning allows models to leverage much larger data sets. In this presentation, we show transfer learning models result in accuracy improvements across a variety of chemical endpoints. The UL Cheminformatics Tool Kit models 74 different chemical endpoints including acute oral-, dermal- and inhalation-toxicity, dermal sensitization, dermal- and eye-irritation, genotoxicity, and acute-and chronic-aquatic toxicity. These models are constructed in two stages. Stage (1) constructs a neural network for each endpoint from chemical structural data. Stage (2) constructs networks with inputs taken from stage 1 hidden layers. We will discuss transfer learning, graph convolutional networks, computational validation approaches and their relevance to chemical hazard modeling.
Date: Friday, May 22, 2020Title: IVAM Virtual Meeting
Kelly Coleman, Phillip Clapp, and Sarah Burnett
Since the 2020 SOT annual meeting was cancelled, IVAM’s leadership decided to have a Virtual Meeting for our members to replace the Reception/Business Meeting that was scheduled for March. The purpose of this meeting is to review IVAM’s activities during the previous year, provide financial and membership updates, plus recognize this year’s IVAM Award winners.
Date: Wednesday, January 29, 2020Title: NAMs Case Study : Industry/Government Partnerships
Monique Perron, ScD
Acting Branch Chief, Risk Assessment Branch IV
US EPA, OPP, HED
Douglas C. Wolf, DVM, PhD, FIATP, ATS
Senior Syngenta Fellow, Product Safety
This webinar will use a case study to discuss the process of leveraging industry/regulatory partnerships to work more efficiently and effectively as we forge ahead in the NAMs space. We will have two speakers, Monique Perron from US EPA, and Doug Wolf from Syngenta. The speakers will discuss the processes they took as they worked through an alternative approach to the 90-day inhalation animal study, highlighting key watch-outs and suggestions from their experience..
Date: January 9, 2020
Abstract: An estimated 3.5-million people die every year from air pollution-induced cardiovascular disease (API-CVD); however, while in vivo data has demonstrated associations between induced CVD and systemic oxidative stress, the causative molecular mechanisms driving API-CVD are still poorly understood. To identify the mechanisms of action driving API-CVD, we developed a unique, tri-culture in vitro model that represents the interface of the alveolar epithelial barrier (AEB) and the cardiovascular system. We hypothesized that air pollutant exposure of human alveolar-like epithelial cells (H441) would induce oxidative stress in adjacent, but physically separated, human pulmonary vasculature cells leading to vascular damage. To test this hypothesis, we exposed confluent, electrically-resistant, and small molecular-impermeable monolayers of H441 cells, grown on the apical surface of Transwell permeable membranes, to the ubiquitous air pollutant, diesel exhaust particulates (DEP), with human lung fibroblasts on the underside of the Transwell membrane and human microvascular lung endothelial cells (HULEC) in the basal compartment. Upon DEP exposure, we identified a variety of oxidative stress responsive genes induced in the directly exposed H441 cells and in the indirectly exposed HULEC, including heme oxygenase 1 (HMOX-1) and NAD(P)H dehydrogenase [quinone] 1 (NQO1). These changes in gene expression occurred despite a lack of change in trans-epithelial electrical resistance (TEER) and small molecule (20kDa fluorescein isothiocyanate (FITC)-dextran) permeability, suggesting that they result from the release of epithelial/fibroblast-derived mediators and/or compounds/metabolites that traverse the epithelial cells. Collectively, these data suggest that induction of oxidative stress in nearby endothelial cells may be mediated by epithelial cells and may be an important mechanism of API-CVD. We conclude that we have developed a relevant, in vivo-like, in vitro model of the AEB-cardiovascular system interface which can be used to identify the elusive molecular mechanisms driving API-CVD. Ultimately, data derived from this model can be used to identify therapeutic targets, biomarkers of both susceptibility and exposure effects, and can encourage the continued development of similar biomimetic models resulting in a reduction of non-essential animal testing.
Abstract: Reliable and effective prediction of hepatotoxicity resulting from repeated exposure to chemicals is of critical importance for the safety assessment of chemicals. However, in vitro long-term repeated exposure studies are difficult to conduct using conventional hepatocyte monolayer cultures due to their rapid de-differentiation and subsequent loss of hepatocyte function. Towards this end, significant advancements have been made to maintain hepatic physiology for an extended period of time by culturing hepatocytes in vitro as three-dimensional (3D) spheroidal aggregates. Here, we compared two promising model systems, HepaRG and primary human hepatocytes (PHH), in both 3D spheroid and traditional 2D monolayer cultures to evaluate their potential as predictive models for assessment of hepatotoxicity. When compared to 2D cultures, cells in spheroid culture maintained higher levels of viability for a longer duration. Likewise, functional characterization revealed higher levels of xenobiotic metabolic activity (CYP1A2, CYP2B6 and CYP3A4) and urea production in spheroid vs 2D cultures. In addition, spheroid cultures showed increased sensitivity to a set of hepatotoxic compounds (ibuprofen, ibufenac, acetaminophen and aflatoxin B1) after repeated exposure (e.g., EC50 of aflatoxin B1 was reduced by 84‰ in PHH and 68 ‰ in HepaRG spheroids as compared to respective 2D cultures), and the sensitivity was further elevated by extended exposure duration (e.g., EC50 of aflatoxin B1 was further reduced by 25 ‰ in PHH and 73 ‰ in HepaRG spheroids when treated for an additional week). Moreover, HepaRG spheroids exhibited similar sensitivity as PHH spheroids especially in extended exposure, suggesting that HepaRG could be a reliable surrogate for PHH and serve as a repeated exposure model to predict hepatotoxicity.
Date: May 9, 2018
Wim H. De Jong, D.V.M., Ph.D., RIVM
Title:Application of the Reconstructed Human Epidermis (RhE) Model as an In Vitro Skin Irritation Test for Detection of Irritant Activity in Medical Device Extracts
Abstract: Assessment of dermal irritation is an essential component of the safety evaluation of medical devices. Testing the irritant capacity of medical device extracts is currently performed by either topical or intradermal injection in rabbits. Attendees will learn about an international validation study that evaluated living human tissues as potential replacements for the rabbit irritation test. In the chemical industry, the RhE model as described in OECD Test Guideline 439 isused to identify irritant chemicals. Although the basic principles remain the same, for application in other domains, adaptation of the assay may be necessary. Therefore, the OECD 439 protocol was modified for testing of medical device extracts. This modified protocol was first evaluated to demonstrate the presence of spiked irritant chemicals added to medical device extracts to show the capability of the modified RhE test protocol to detect irritants in a complex extract mixture. The real proof of the test would be to detect irritants in extracts of medical devices or materials used in the production of medical devices. Therefore, specific polymer materials were prepared containing irritant chemicals within the polymer matrix. With these materials two RhE models, EpiDerm™ (MatTek, Inc.) and SkinEthicTM RHE (EpiSkin, SA), were evaluated in an international round robin study. The read-out endpoint was the same as in OECD 439, that being tissue viability by the MTT method; in addition, Interleukin 1a release was also considered. In order to enhance the impact and future implementation of the RhE assay for medical device safety testing, a broad range of stakeholders were encouraged to join the round robin. Participants included medical device companies, contract research organizations, university and governmental laboratories. The results of the round robin study showed that RhE tissue models could detect the presence of strong skin irritants at low levels in dilute medical device polymer extracts. Consequently, the protocol will now be adapted and proposed as a new ISO 10993 standard for medical device irritation testing.
Abstract: An underlying limitation in the adoption of in vitro and alternative testing strategies has been the lack of consensus in how best to evaluate the predictivity of these approaches for use in chemical safety assessments. Significant advances in experimental technologies and computational approaches over the past ten years have provided the data necessary to initiate several follow-up efforts to start providing such an evaluation. This webinar will provide perspectives of three scientists with first-hand experience in these assessments, covering governmental and industry efforts as well as national and international perspectives.
Date: Wednesday, June 7, 2017
Louis Scarano, PhD, US Environmental Protection Agency
Title: TSCA as Amended by the Frank R. Lautenberg Chemical Safety for the 21st Century Act: The New Future for Alternative Test Methods
Abstract: This presentation will present the current regulatory landscape of alternative test methods and industrial chemicals in the US. The US EPA Office of Pollution Prevention and Toxics (OPPT) is responsible for evaluating and regulating industrial chemicals in the US. In evaluating new chemicals, OPPT extensively uses alternative methods (i.e., QSAR, read-across/analog/category approaches, tiered testing, etc.) to determine hazard/risk. For existing chemicals, evaluation generally relies on conventional in vivo studies and category/read across where possible. The Frank R. Lautenberg Chemical Safety for the 21st Century Act was signed into law on June 22, 2016. It amended and updated the 1976 Toxic Substances Control Act (TSCA). Section 4(h)(1) states that “The Administrator shall reduce and replace, to the extent practicable, scientifically justified, and consistent with the policies of this title, the use of vertebrate animals in the testing of chemical substances or mixtures.” US EPA is required in Section 4(h)(2)(A) to “develop a strategic plan to promote the development and implementation of alternative test methods.” by June of 2018.
Kate Willett, PhD, Humane Society of the United States
Title: The Role of Non-Animal Safety Assessment Methods in Implementation of the New TSCA
Abstract: The Frank R. Lautenberg Chemical Safety for the 21st Century Act was signed into law by President Obama and came into force on June 22, 2016. This amendment to the Toxic Substances Control Act (TSCA) increases the authority of the US Environmental Protection Agency (EPA) to obtain information on both new and existing industrial chemicals and includes a mandate to both use the “best available science” and to minimize vertebrate testing. Under the amended TSCA, existing chemicals will be subject to prioritization via a risk-based screening process into high and low priority. The amended Act is therefore likely to require the generation of massive amounts of new information on both existing and new industrial chemicals and also provides an opportunity for US EPA to take advantage of recent advances in testing approaches focused on providing adequate information while minimizing animal testing for both prioritization and risk assessment, such as: 1) existing replacement methods; 2) risk-based decision matrices (e.g., RISK21); 3) adverse outcome pathway-supported integrated approaches to testing and assessment; 4) new high-throughput testing approaches (e.g., ToxCast); and 5) greatly improved hazard and exposure estimation modeling. At the same time there is pressure to reduce vertebrate testing, creating an immediate need for increased implementation of non-vertebrate evaluation tools. This talk will describe US EPA’s proposals for prioritization and risk assessment and suggest recommendations for adapting these proposals to meet both regulatory and statutory requirements.
Date: May 18, 2017
Fabian Grimm, PhD, Texas A&M University
Title: Diversity In a Dish: A Human Population-Based Organotypic In Vitro Model for Cardiotoxicity Testing
Abstract: Recent advancements in stem cell technologies have opened the opportunity to generate virtually unlimited numbers of genetically-defined, donor-specific induced pluripotent stem cell (iPSC)-derived cell types. Dr. Grimm’s presentation will exemplify how a human population-based in vitro cardiotoxicity model can be used to inform chemical safety testing and human health assessments. This model is amenable for (1) providing chemical-specific uncertainty factors for estimation of inter-individual susceptibilities to adverse chemical effects and (2) extrapolation of in vitro-derived dose-response relationships to physiologically-relevant exposure levels.
Rachel Shaffer, University of Washington
Title: Lifestage-Specific Modeling Platform for Adverse Outcome Pathways of Male Reproductive and Developmental Processes
Abstract: Early life development of the male reproductive system is vulnerable to disruption from chemical exposure, particularly during critical windows of susceptibility, yet robust alternative models are lacking. This webinar will highlight research from the Faustman lab at the University of Washington-Seattle to 1) Develop a systems biology platform for integrating normal and adverse responses across testis development in rodents in vivo and in vitro; 2) Establish a new mouse co-culture and quantify baseline characteristics of the system; 3) Use the mouse co-culture to evaluate effects of cadmium treatment during critical windows of susceptibility.
Johanna Nyffeler, University of Konstanz
Title: Development and Application of an In Vitro Human Neural Crest Cell Migration Assay
Abstract: Neural crest cells (NCCs) are a transient stem cell population arising at the time of neurulation. NCCs migrate to various body parts and differentiate in cells of the peripheral nervous system, melanocytes as well as craniofacial bones and other structures. Therefore, failure of NCC to migrate can lead to severe developmental defects, so called neurocristopathies.
We developed an in vitro test system to screen compounds for interference with human NCC migration. From the compounds tested so far (> 100 compounds), several pesticides, flame retardants, halogenated compounds and few drug-like compounds interfered with NCC migration at concentrations that were not cytotoxic. The test system could be used in a test battery to prioritize chemicals for more complex, lower throughput developmental toxicity studies.
Date: Monday, April 10, 2017
Kim Li, PhD, DABT, MPH, Amgen Inc.
Title: ISO 10993-1 Biological Evaluation—The Risk Management of Unstudied Extractables and Leachables (E&L) Impurities in Medical Devices and Combination Products
Abstract: Dr. Li’s presentation will show how the integration of in silico modelling into a toxicology assessment can be useful in the screening of the unstudied extractables and leachables impurities from medical devices. She will include a discussion of the framework described in the US Food and Drug Administration's guidance on the use of ISO 10993-1 “Biological evaluation of medical devices—Part 1: Evaluation and testing within a risk management process” (2016). The presentation will highlight the three different modules of Toxtree (IdeaConsult)—in vitro mutagenicity (Ames test) prediction, Cramer classification for systemic toxicity, and protein binding alerts—which can offer understanding of a substance’s chemical reactivity and toxicity, and potentially replace certain biocompatibility tests.
Marco Viceconti, PhD, Executive Director, Insigneo Institute for In Silico Medicine, the University of Sheffield and Sheffield Teaching Hospital NHS Foundation Trust
Title: Animal-Specific Modelling for the 3Rs in Pre-Clinical Assessment: A Bone Drugs Example
Abstract: Dr. Viceconti’s presentation will include a case study comparing the results of experiments in mice to test the efficacy of bone drugs with an animal-specific modelling approach based on longitudinal imaging. Initial results demonstrated that the in silico modelling approach could reduce the number of animal used by 63 ‰, and suggest that inclusion of human-specific modelling may improve the ability to predict safety and efficacy of new drugs before they are tested on humans.
Abstract: Many graduate students are interested in furthering the field of alternatives to animal testing, but do not know what career options are available to them or how to prepare for such a career. With this in mind, the In Vitro and Alternative Methods Specialty Section and Graduate Student Leadership Committee are teaming up to provide this information to students interested in pursuing a career focused on alternatives to animal testing.
Jenna Currier, PhD, US EPA
Title: What’s in a Tipping Point? Using Systems Biology to Characterize Adaptive and Adverse Oxidative Responses in Human Lung Cells
Abstract: Implementing high-throughput screening paradigms and risk assessment based on in vitro and in silico testing requires utilizing toxicity pathway information to distinguish adverse outcomes from recoverable adaptive events. However, little work has focused on oxidative stresses in human airway for the purposes of predicting adverse responses. This talk highlights research investigating molecular mechanisms at the tipping point of adaptive and adverse responses in human bronchial epithelial cells exposed to zinc, a model oxidant.
Advisor: Brian Chorley, PhD
Shih-Yu (Shirley) Chang, MS, PhC, University of Washington
Title: Proof of Concept Study—Microphysiological Systems (MPS) to Identify Organ-Organ Interactions in Toxicology: Hepatic Metabolism Enhances Nephrotoxicity of Aristolochic Acid
Abstract: MPS represent an interconnected set of cellular constructs designed to recapitulate the structure and function of human organs. MPS are also frequently referred to as organs-on-chips or in vitro organ constructs. Once validated, well-designed in vitro studies utilizing such systems could be transformative for future predictive toxicology studies. Although much work has been accomplished with individual organ-based MPS models, there has been relatively little achieved in utilizing combined organ MPS approaches to identify toxicologically relevant organ-organ interactions in vitro. This talk highlights some of the results from University of Washington’s collaborative research in liver-kidney-chip model to evaluate the toxicological impact of hepatic metabolism of Aristolochic Acid (AA), a well-known nephrotoxin and carcinogen, on its nephrotoxicity.
Advisor: David Eaton, PhD
Kate Willett, Director Regulatory Toxicology, Risk Assessment and Alternatives, Humane Society of the United States
Title: Introduction to the Concept of Adverse Outcome Pathways and IATA according to the Organization of Economic Cooperation and Development
Anna Lowit, Senior Scientist, Office of Pesticide Programs, US Environmental Protection Agency
Title: Practical Application of IATA including Non-Standard Test Methods
Abstract: Along with the availability of an increasing number and variety of methodologies to assess chemical activity is a consequent need to assess their reliability and utility efficiently, with the goal of efficient and practical utility for decision-making. It is also clear that these methods will necessitate the use of a combination of existing information, strategic testing and prediction models. Adverse Outcome Pathways can form the logical basis for the integration of information and tests into integrated approaches to testing and assessment (IATA) in the context of exposure and specific regulatory application. The Organization for Economic Cooperation and Development (OECD) has made a significant investment in development of AOPs, and is currently developing guidance for IATA. OECD is also developing guidance to streamline validation of these novel technologies. The presentations will include examples using pesticide classes to highlight the value of IATA to regulatory testing and risk assessment.
Andries van der Meer, PhD, Wyss Institutefor Biologically Inspired Engineering at Harvard University
Title: Human Organs-on-Chips as Replacements for Animal Testing
Introduction by IVAM Past President Suzanne Fitzpatrick, PhD, US FDA
Title: Advancing FDA Regulatory Science Through Innovation—In Vitro Microphysiological Systems
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.