November 27, 2023 10:00 AM – 11:00 AM EDT / 16:00 – 17:00 CET

Register here: https://us06web.zoom.us/webinar/register/WN_HKJ5RrWLS4atk72Xwv5nxg#/registration

Presenter: Jo Nyffeler, PhD, Helmholtz Centre for Environmental Research (UFZ)

“Cell Painting” is an imaging-based, high-throughput method to obtain phenotypic information of individual cells. Initially used mainly in pharma industry for hit discovery, Cell Painting can be applied to other research questions, such as safety assessment.
The Center for Computational Toxicology and Exposure at the U.S. Environmental Protection Agency has applied Cell Painting to environmental chemicals and explored its use for screening-level chemical hazard evaluation of environmental chemicals.

In this webinar, I will walk you through the findings of our recent publication (Nyffeler et al. 2023, PMID: 37044265, DOI: 10.1016/j.taap.2023.116513). Using multiple examples, I will outline the applicability of Cell Painting. Cell Painting can be used to obtain in vitro potency estimates for chemicals that can be compared to exposure information. Moreover, as others have already shown, Cell Painting can be used to group chemicals with similar mode-of-actions. We showed that this also works for environmental chemicals with less defined mechanisms-of-action. Cell Painting is able to also highlight critical differences that would not have been apparent when only looking at structural information. Thus, this webinar will give you an overview of possible application of Cell Painting in the context of safety assessment.

Tuesday, November 14, 2023 10:00 AM – 11:00 AM EDT / 16:00 – 17:00 CET

Register here: https://us06web.zoom.us/webinar/register/WN_ikYuOLk4QAqFCcJnR0_kbg#/registration

Monday, October 30, 2023 9:00 AM – 11:00 AM EDT  / 14:00 -16:00 CET

Register here: https://us06web.zoom.us/webinar/register/WN_9nSZrHAwS-uSE954Mni-7A#/registration

Presenters:Olga Tcheremenskaia, PhD, Istituto Superiore di Sanità (ISS)
Andrea Gissi, PhD, European Chemicals Agency (ECHA)
Patience Browne, PhD, Organisation for Economic Co-operation and Development (OECD)

This webinar will provide an overview new OECD (Quantitative) Structure-Activity Relationship ((Q)SAR) Assessment Framework (QAF) which is a systematic and harmonised framework for the regulatory assessment of (Q)SAR models, predictions, and results based on multiple predictions. The framework includes new principles for the assessment of (Q)SAR predictions and results, a checklist of criteria, along with guidance for assessing each criterion, to help establish confidence in the use of (Q)SARs in evaluating chemical safety.  The QAF is meant to be applicable irrespective of the modelling technique used to build the model, the predicted endpoint, and the intended regulatory purpose. The primary audience of this document is regulatory authorities and their stakeholders. In addition, any other (Q)SAR user is encouraged to refer to the QAF when using (Q)SARs for regulatory purposes.

The webinar will start with an overview of the project. Then, two talks will cover the main aspects of the framework, namely the assessment of models and of results based on individual or multiple predictions. The presentations will also include example of assessment according to the checklists part of the OECD document.

Recordings and other materials from this webinar will be posted on the ASCCT webinar archive shortly after the webinar’s close.

The recording is available here: https://www.youtube.com/watch?v=wRX-nU3oacg

Human biomonitoring to support risk assessment and chemical policies in Europe: results from HBM4EU 

Presented by Greta Schoeters, PhD

Human biomonitoring (HBM) measures chemicals or their metabolites directly in human tissues or fluids and hence aggregates chemical exposure from different sources and intake routes. As such it fits with the one chemical- one assessment concept which is part of Europe’s strategy for sustainable use of chemicals. Human biomonitoring provides a picture of reallife exposure to chemicals which is essential information for hazard characterisation and chemical risk assessment. A coherent human biomonitoring framework is still lacking in Europe. However, it is needed to track the progress towards EU’s Zero Pollution Action which aims to reduce pollution levels to be no more harmful for health by 2050. The European project HBM4EU (2017-2022) operated at the science policy interface and has established a European Union-wide HBM programme to generate knowledge on human internal exposure to chemical pollutants and their potential health impacts in Europe.  Priority substance groups were identified to meet the most important needs of both European and national policy makers and risk assessors.  Building on existing capacities, HBM4EU brought together scientists from 30 countries. As one of the core activities, harmonized human biomonitoring data from different European regions and age groups were collected. The HBM4EU Aligned Studies generated new HBM data of 10,795 participants. HBM4EU has set up a laboratory network to produce comparable high quality analytical data, provided wide access to data and facilitated interpretation of results by developing indicators and human biomonitoring guidance values.   

Results displayed the heterogeneity of internal chemical exposure of European residents: geographically, according to the age group, according to the sampling period, according to the educational level of study participants. The levels of some substances in the human body of the European population are still so high that adverse health effects cannot be excluded according to current knowledge. 

HBM4EU produced data that can serve as a baseline to evaluate the success of the measures taken to operationalise the Chemicals Strategy for Sustainability and the Zero Pollution Agenda in the frame of the European Green Deal, it signaled upcoming substances that are present in the body. HBM4EU demonstrated that not only regulations but clear strategies are needed to prevent further pollution on the human body. 

Human biomonitoring for the identification of real-life chemical mixtures 

Presented by Mirjam Luijten, PhD 

Human biomonitoring (HBM) approaches are valuable for chemical risk assessment, since they allow for measurements of chemicals or their metabolites directly in human tissues or fluids and hence aggregates chemical exposure from different sources and intake routes. As such, HBM data are also highly valuable for mixture risk assessment. Network analysis applied to HBM data may provide insight into real-life mixtures by visualizing chemical exposure patterns. The identification of groups of more densely correlated biomarkers, so-called “communities”, within these networks highlights which combination of substances should be considered in terms of real-life mixtures to which a population is exposed. We applied network analyses to HBM datasets from four different countries. Our approach demonstrates that network analysis applied to HBM data of highly varying origin provides useful information with regards to the existence of groups of biomarkers that are densely correlated. The next questions are whether the combined body burden of multiple chemicals is of potential health concern. If so, subsequent questions are which chemicals and which co-occurrence patterns are driving the potential health risks. To address this, we developed a biomonitoring hazard index. Our analysis revealed that this biological index method can put forward communities of co-occurrence patterns of chemicals on a population level that need further assessment in toxicology or health effects studies.  

Another valuable approach for mixture risk assessment is suspect screening. Within HBM4EU a  harmonized suspect screening approach was developed, aimed at the identification of new exposure biomarkers for pesticides. This approach was used in a suspect-screening based study to describe the probability of (concomitant) exposure to a set of pesticide profiles in five European countries We explored whether living in an agricultural area (compared to living in a peri-urban area), being a a child (compared to being an adult), and the season in which the urine sample was collected had an impact on the probability of detection of pesticides. Forty pesticide biomarkers relating to 29 pesticides were identified at high levels of confidence in samples across all study sites. We observed differences in the probability of detection of a pesticide (metabolite) among children compared to adults, but not for area or season. This survey demonstrates the feasibility of conducting a harmonized pan-European sample collection combined with suspect screening to provide insight into the presence of exposure to pesticide mixtures in the European population.  

Taken together, our results from HBM4EU demonstrate the usefulness of HBM data for human health risk assessment of chemical mixtures.  

Wednesday, May 24, 2023 10:30 AM – 12:00 PM EDT/ 16:30 – 18:00 CET

Liver spheroids as an in vivo like human in vitro system for prediction of NASH, drug metabolism and drug-induced hepatotoxicity

Presented by Magnus Ingelman-Sundberg, PhD, BSc.Med

Drug development is a challenging pursuit, with over 85% of drugs failing during clinical development due to inadequate preclinical systems for compound selection. Liver toxicity is a significant contributor to these safety failures, and despite numerous attempts to develop pharmacological therapies for chronic liver diseases, including NASH and fibrosis, success has remained elusive. Given the costs and patient burden associated with clinical trials, novel strategies are urgently needed to improve success rates. One promising approach involves using human liver spheroids, which can maintain patient-specific phenotypes and functions for several weeks in culture. In this lecture I explore recent applications of liver spheroids for analyzing drug-induced liver toxicity, evaluating the disposition and metabolism of low clearance drugs, and developing treatments for metabolic and infectious liver diseases. With increased use, liver spheroids have the potential to become the new gold standard in translational pharmacology and toxicology.

In vitro prediction of liver toxicity using adverse outcome pathways

Presented by Mathieu Vinken, PhD, PharmD, ERT

Adverse outcome pathways (AOP) and their networks are tools to capture and visualize mechanisms of toxicity. A multitude of AOPs and AOP networks related to liver toxicity has been introduced over the past decade. This is not a surprise, as the liver is the poison control center of the body due to its unique location and function. The present webinar will focus on the development and practical validation of an AOP network mechanistically describing the accumulation of noxious bile acids in the liver or so-called cholestasis induced by chemical compounds, including pharmaceutical drugs, food additives and cosmetic ingredients. It will be demonstrated how this AOP network can serve as the conceptual basis for reliable in vitro detection and prediction of chemical-induced cholestatic liver injury

Friday, April 28, 10:00 – 11:00 ET/ 16-17:00 CET
The recording is available here: https://www.youtube.com/watch?v=yYcgSB53t38

Development of an adverse outcome pathway for kidney tubular necrosis

Presented by Devon Barnes, Utrecht Institute for Pharmaceutical Sciences

Adverse outcome pathway (AOP) networks combine AOPs that share one or more key events (KEs). Our aim is to develop an AOP network determining KEs and the relationships that drive chemical-induced kidney tubular necrosis (TN). To weigh the evidence between KEs, the network will be assessed in accordance with guidelines from the Organization for Economic Co-operation and Development (OECD). The objective is to develop an ontological knowledge framework that integrates biological, toxicological, and chemical data toward predicting systemic repeated dose toxicity effects of nephrotoxic chemicals associated with kidney TN.

Embase was used to search for literature on chemical-induced kidney TN using key search terms relevant to clinical presentations, biochemistry, histology, and chemically applicable, data-rich nephrotoxic compounds. Initial title/abstract screening of papers employed SysRev, a computational tool for systematic reviewing and data extraction, using labeling strategies for inclusion/exclusion criteria. Tailored Bradford-Hill criteria described in OECD guidelines will assess confidence levels and weight of evidence for KEs within the AOP network. Kidney physiological maps were designed to establish mechanisms contributing to TN, with systemic mapping of currently reported AOPs involving nephrotoxicity identifying relevant MIEs and KEs.

The Embase search retrieved 2735 papers to upload to SysRev. The title/abstract screening would further identify papers eligible for data extraction in the full-text screening process. A total of 19 existing AOPs related to kidney dysfunction were identified and analyzed to support the implementation of additional in vitro endpoints for TN.

Data extracted will assess confidence levels in previously described KEs and KERs and identify potential new KEs. The AOP network will form the conceptual basis for establishing a test battery of in vitro assays to characterize nephrotoxic chemicals by measuring individual KEs for the generation and evaluation of AOPs of TN-related kidney failure.

Computational modelling of neural tube closure defects

Presented by Job Berkhout, RIVM

Closure of the caudal neural tube is a critical event that occurs early in development, around day 27 of human gestation. Failure of neural tube closure results in severe birth defects, such as spina bifida. These neural tube defects (NTD) are among the most prevalent human congenital malformations, which warrants specific attention in chemical safety assessment. Computational models of biological processes are likely to revolutionize chemical safety assessment in the near future. Such models can be used to predict the effect of chemical-induced gene expression changes and provide a template for establishing quantitative adverse outcome pathway networks. This study aims to develop an in silico model of the human neural tube closure, which will be applied to predict chemical-induced NTDs.

By extensively mining the developmental biology and toxicology literature, we first created a physiological map of human neural tube closure. Based on the physiological map, we built a multicellular agent-based model using CompuCell3D. The constructed physiological map depicts the all-trans-retinoic acid (ATRA) related molecular pathways linked to the various cell types in which they occur, and their morphogenetic consequences, that lead to closure of the neural tube. The morphogenetic events driven by gene expression changes are visualized by the computational model.

We simulated in silico the complex biological process of neural tube closure, in order to demonstrate the feasibility of this approach. At a later stage in the project, the computational model will be applied to predict chemical-induced changes in gene expression and cell characteristics. The predictions of the model will be validated using a set of dedicated in vitro assays in conjunction with existing knowledge on in vivo developmental neurotoxicity. Such computational models may ultimately provide an alternative in silico approach for chemical safety assessment without the use of animals.

Friday, March 24, 2023, 10:00 – 11:00 a.m. ET / 16:00 -17:00 CET

The recording is not available

A multiorgan-on-chip platform for the in vitro investigation of off-target cardiotoxicity of liver-metabolized anticancer drugs

Presented by Erika Ferrari, PhD

Off-target cardiotoxicity is one of the main causes of drug withdrawal from the market. Multiorgan-on-chip (MOoC) platforms represent a disruptive solution to predict liver metabolism on off-target organs to ultimately improve drug safety testing during drug development. Here we integrate liver and cardiac models in a compartmentalized valve-based MOoC and we show its application in studying the effects of liver-metabolized Terfenadine on cardiac microtissues. The two-compartment MOoC integrates a liver micropatterned co-culture (MPCC) of HepG2 and fibroblasts with a 3D mechanically stimulated cardiac microtissue generated from neonatal rat cardiomyocytes. Communication between compartments is achieved through a system of normally-closed valves, whose aperture is controlled via an overlaying vacuum-activated control layer. Numerical and experimental simulations were conducted to evaluate the dynamics of drug diffusion across the compartments. Medium supplemented with 10μM Terfenadine was administered to the liver; once metabolized (after 24h), cardiac microtissue viability and functionality were assessed by means of an integrated electrical recording system upon valve aperture. The platform was validated for pharmacokinetic-based drug screenings by measuring the effect of the anticancer drug Terfenadine after metabolism by MPCCs in the liver compartment. While non-metabolized Terfenadine caused a significant decrease in cardiac cell viability and an increase in field potential duration (FDP), its non-toxic metabolite Fexofenadine did not cause any significant alteration in cardiac microtissues. We developed a valve-based MOoC for liver-heart compartmentalized cultures. The system allows for controlled diffusion of liver-metabolized drugs (e.g., Terfenadine) to a cardiac compartment while excluding cell-cell interactions and eliminating convective transport, demonstrating the potential for studying drugs off-target cardiotoxicity upon liver metabolism.

Predicting developmental toxicity of pyrethroid insecticides in vitro using human-induced pluripotent stem cells 

Presented by Yanying Ma

Metabolites of pyrethroid insecticides are detected in most urine samples from the general population. Pyrethroids act primarily by interfering with voltage-gated sodium channels, which are essential for nerve cell function and cardiac muscle contraction. Therefore, pyrethroids are suspected to be cardiotoxic. Furthermore, pyrethroids are suggested to interfere with thyroid hormones that are essential for foetal brain development. Thus, exposure to pyrethroids during vulnerable windows in pregnancy may adversely affect child neurodevelopment and cardiovascular health. The adverse effects of three pyrethroid insecticides (deltamethrin, α-cypermethrin and etofenprox) and the common metabolite 3-phenoxybenzoic acid (3-PBA) on cardiomyocyte differentiation were assessed in an in vitro 3D model of human induced pluripotent stem cell-derived embryoid bodies (EBs) that mimic the very early stage of the human embryo – the blastocyst. A cardiac-specific reporter gene assay – the PluriLum, was used as a cardio-developmental toxicity endpoint. The binding ability of pyrethroids to transthyretin (TTR) was assessed using the cell-free ANSA-TTR binding assay. The EBs were differentiated into cardiomyocytes and the effects of the pyrethroids on this process was measured. Deltamethrin, α-cypermethrin and etofenprox had a significant negative impact on differentiation (LOECs: 13, 6.3 and 1.6 μM, respectively). 3-PBA showed no effect at any tested concentrations (up to 100 μM). However, it significantly displaced ANSA from TTR (LOEC = 1.6 μM), whereas the parent pyrethroids showed no TTR-binding potential at the range of tested concentrations (up to 200 μM). Our results indicate that pyrethroid parent compounds have the potential to adversely affect cardiac differentiation. On the other hand, only the metabolite, 3-PBA, could bind to TTR and potentially disturb thyroid hormone transportation. These findings highlight the risks posed by insecticides on human foetal development, supporting the need to restrict their use in order to reduce human exposure.

Wednesday, February 22 at 11:00am – 12:30pm ET /  16:00 -17:30 CET
The recording is available here:https://www.youtube.com/watch?v=FTpgjMcCc1k&t=2s

Integrating High Throughput Transcriptomics into a Tiered Framework to Prioritize Chemicals for Toxicity Testing 

Presented by Jesse Rogers, PhD

US EPA is developing a tiered assessment strategy for chemical toxicity testing by integrating multiple data streams. Pairing high content assays such as high-throughput transcriptomics (HTTr) with high-throughput screening (HTS) of specific molecular targets may improve confidence when assessing key hazards. Here, we used HTTr screening data to generate new signatures representing known molecular targets, and signature-level potency estimates were integrated with orthogonal HTS assays as a proof-of-concept framework for chemical prioritization.

Transcriptomic profiles generated via the TempO-Seq platform in both HepaRG and U-2 OS cell lines were used to develop signatures comprised of genes selectively responsive to reference chemicals for one of 13 distinct molecular targets. Of 1,218 chemicals screened in HTTr to date, 232 chemicals demonstrated selective potency in at least one reference signature versus non-selective potency estimates. In examining these chemicals using available orthogonal HTS assays from US EPA’s ToxCast program, 74 chemicals were confirmed as selective AHR, GR, or RAR/RXR nuclear receptor agonists.

Our work demonstrates that HTTr data can inform putative molecular targets and identify chemicals for further screening in a framework to support chemical risk assessment.

The views expressed in this abstract are those of the authors and do not necessarily reflect the views or policies of the US EPA.

Evaluation of Per- and Poly fluoroalkyl Substances (PFAS) in vitro toxicity testing for developmental neurotoxicity

Presented by Kelly Carstens, PhD

Per- and poly fluoroalkyl substances (PFAS) are a diverse set of commercial chemicals widely detected in humans and the environment. However, only a limited number of PFAS are associated with epidemiological or experimental data for hazard identification. To provide developmental neurotoxicity (DNT) hazard information, the work herein employed DNT new approach methods (NAMs) to generate in vitro screening data for a set of 160 PFAS. The DNT NAMs battery was comprised of the microelectrode array neuronal network formation assay (NFA) and high-content imaging (HCI) assays to evaluate proliferation, apoptosis, and neurite outgrowth. The majority of PFAS (118/160) were inactive or equivocal in the DNT NAMs, leaving 42 active PFAS that decreased measures of neural network connectivity and neurite length. Analytical quality control indicated 43/118 inactive PFAS samples and 10/42 active PFAS samples were degraded; as such, careful interpretation is required as some negatives may have been due to loss of the parent PFAS, and some actives may have resulted from a mixture of parent and/or degradants of PFAS. PFAS containing a perfluorinated carbon (C) chain length ≥8, a high C:fluorine ratio, or a carboxylic acid moiety were more likely to be bioactive in the DNT NAMs. Of the PFAS positives in DNT NAMs, 85% were also active in other EPA ToxCast assays, whereas 79% of PFAS inactives in the DNT NAMs were active in other assays. These data demonstrate that a subset of PFAS perturb neurodevelopmental processes in vitro and suggest focusing future studies of DNT on PFAS with certain structural feature descriptors. (This abstract does not reflect U.S. EPA Policy).

A Modern Framework to Establish Scientific Confidence in New Methods

Presented by Anna van der Zalm, MChem

Current processes to validate new non-animal methods are costly, time-consuming, and do not necessarily produce methods that are fit for regulatory purposes. This talk will build on previous efforts from the Organisation for Economic Co-operation and Development and the International Cooperation on Alternative Test Methods to propose a modern, flexible framework comprising five essential elements to establish scientific confidence in non-animal methods for regulatory use: fitness for purpose, human biological relevance, technical characterization, data integrity and transparency, and independent review.

Updates to the current process are based on the recognition that (a) the relevance of the results of the new method need not be determined through direct alignment with the results of the traditional animal test method, and instead may be determined through alignment with, or fidelity to, human biological understanding; (b) the new method should not be required to replace the traditional animal test method one to one, nor produce the same information generated by the traditional animal test method; (c) the currently accepted levels of reproducibility in traditional animal test methods can be used to inform performance benchmarks for new methods; (d) ring trials may not be necessary for the assessment of the reproducibility of a new method; and (e) preferably before a method is developed, its purpose should be clearly defined and discussed amongst the method developer, regulators, and the regulated industry to ensure the production of new non-animal methods that are fit for purpose.

Thursday, January 19 at 10:00 – 11:00 a.m. ET
The recording is available here: https://www.youtube.com/watch?v=cdTrot93feA&t=3s

Facilitating Global Connections through the Microphysiological Systems for COVID Research (MPSCoRe) Working Group

Presented by Amber Daniel, M.S.

The emergence and global spread of the coronavirus disease 2019 (COVID-19) emphasizes the need for effective approaches to prevent, control, and treat infectious diseases. Although animal models have historically been used to address such challenges, human cell-based in vitro platforms known as microphysiological systems (MPS) have the potential to more efficiently and effectively model the human lung and other organ systems affected by COVID-19. However, absent a venue for coordination, widespread application of MPS to COVID-19 research presents a risk of overlapping investigations and duplication of efforts among researchers. The MPS for COVID Research (MPSCoRe) working group was organized to reduce this risk by globally connecting key MPS stakeholders from the research, method development, drug and vaccine manufacturing, and regulatory sectors. The working group facilitates open communication among stakeholders to maximize the impact of MPS technologies in understanding disease mechanisms and treatments, and reducing animal use while improving human health. In this way, the group aims to promote adoption of MPS for studying COVID-19 and future emerging infectious diseases. Activities currently supported by MPSCoRe include a proof-of-concept study to evaluate MPS models for testing the safety and efficacy of novel COVID-19 therapeutics. Additionally, MPSCoRe is supporting development of a web-based repository for sharing COVID-19 experimental data and other MPS resources. These efforts will accelerate the development and adoption of MPS in infectious disease research, thereby reducing the reliance on animal models in this space. This project was funded with federal funds from NIEHS, NIH under Contract No. HHSN273201500010C.

Applying Deep Learning Toxicity Models Across the Chemical Universe

Sunggun Lee

Chemicals are ubiquitously found in many exposure routes in day-to-day life. It is important that toxicity testing guides maximally informed decisions on chemical use and regulation to best protect human health. Traditional toxicity testing generally relies on in vivo or in vitro methods which are time-consuming, resource-intensive, and frequently results in flawed generalizations to humans. Collaborators at the FDA/NCTR developed two novel deep learning models called DeepCarc and DeepDILI to predict endpoints for carcinogenicity and drug-induced liver injury, respectively, outperforming state-of-the-art methods. These deep learning models incorporate an ensemble approach of five conventional machine learning algorithms integrated into a neural network to generate probabilistic predictions for carcinogenicity and liver injury. Here, DeepCarc and DeepDILI were reproduced and refined, and the externally validated models were applied to screen the carcinogenicity and liver injury potential of 7176 compounds in the Tox21 database. The majority of the Tox21 compounds were predicted to have low carcinogenicity concern with only 88 of the compounds possessing a carcinogenicity probability of greater than 0.9. DeepDILI predictions suggested that the compounds in Tox21 generally have a higher risk for liver injury with many compounds predicted to have a liver injury probability of greater than 0.9. Compounds with high toxicity probability predictions were run through Integrated Chemical Environment Characterization workflows to investigate the distribution of physicochemical properties, presence in consumer products, and bioactivity profiles from high-throughput screening assays. Such computational models can be used to rapidly screen large chemical libraries to prioritize potentially hazardous substances for further examination.

December 1 at 9:00 – 10:00 a.m. EST / 15:00 – 16:00 CET

PRIVAT: A tool for improving the peer-review of in vitro studies
Paul Whaley, PhD
Achieving consistent, thorough peer-review of in vitro studies in a way that provides transparent, actionable information for editorial decision-making is very challenging. To help with this, a new tool (PRIVAT) is being developed by the Evidence-Based Toxicology Collaboration. This presentation will discuss the unusually rigorous methods behind ensuring the tool is a genuine help to reviewers and editors, rather than just another questionnaire that complicates the peer-review process without obviously improving it. These methods include a systematic review of existing tools (yielding over 700 unique quality criteria) and a Delphi process that whittled the large number of criteria down into a meaningfully applicable set of questions and quality judgements. Prospects for uptake of the tool and the general challenges of improving publishing standards in toxicology, including ensuring data availability, will also be discussed.

Increasing reliability of science with open and dynamic methods in academic publications
Sofia Batista Leite, PhD
Clear reporting of scientific methods and protocols in academic publications is the basis for reproducible, reliable and progressive science. Open data has recently received a great deal of attention, but in reality, the usability of data is only ensured when the methods for generating it are clearly described.  Making progress  towards open, detailed and dynamic protocols in peer reviewed publications requires  huge investment from all of the different agents in the publication system, from researchers to publishers, the institutions in which they work and the funders that support them. A concerted community effort by all of these different agents is needed in order to create a culture that embraces, supports and rewards good method reporting. With prior experience in the acceptance of methods and data for regulatory purposes, the European Commission Joint Research Centre convened a group of scientists and publishers to undertake the translation of discussion about making methods more clearly reported in life science publications into clear action points. In this presentation, I will talk about the main actions to be undertaken by the key actors in research institutions, funding bodies and publishers that can trigger and support the needed culture change. The work will soon be open for consultation. With this work, we expect to support more transparent, reliable and robust science, of a higher quality and more impact.   
On the top of increasing the quality and impact of scientific research we believe that this can facilitate the acceptances of new methods in the regulatory field. This activity promotes clarity to build trust in non-guideline methods in support of the use of academic data for regulatory purposes.

September 30 at 10:00-11:30am EDT/14:00-15:00 CET

Expectations for new approach methods performance in predicting effects in repeat dose animal studies
Katie Paul Friedman, PhD, Center for Computational Toxicology and Exposure, Office of Research and Development, US EPA
Building scientific confidence in new approach methodologies (NAMs) for regulatory toxicology may include a comparison to in vivo results. This presentation overviews work to characterize benchmark expectations for NAM performance in predicting systemic and organ-level effects in repeat dose studies of adult animals using data curated into the Toxicity Reference Database. The reproducibility, variability, and differences in systemic and organ-level findings (liver, kidney, stomach, spleen, thyroid, and adrenal) in repeat dose studies, using lowest effect level (LELs) for body and/or organ weight, as well as gross and/or histopathological changes, were investigated.
Quantitative variability in LELs was examined with multilinear regression modeling, using study descriptors as covariates to estimate total variance, mean square error (MSE), and root residual mean square error (RMSE) in systemic and organ LELs. The MSE values suggest study descriptors accounted up to 70% of total variance in organ and systemic LELs, and that RMSE ranged 0.4 – 0.6 log10-mg/kg/day.
A quantitative structure activity relationship model for repeat dose point-of-departure (POD) estimates was constructed that used this RMSE estimate of variability to construct a POD distribution and 95% confidence interval for each POD prediction.
Qualitative variability was further examined. The concordance of organ-level findings in replicate studies, defined by chemical only, chemical and species, and chemical and study type, ranged 39 – 88%. Odds ratios indicated it is unlikely to observe organ effects in a chronic study if the chemical was negative in a subchronic study.
Overall, quantitative and qualitative variability in repeat dose LEL values suggests that a good NAM might predict systemic or organ LELs within ± 1 log10-mg/kg/day with accuracy approaching 70%. Organ level findings are most reproducible within-species, and a subchronic study is likely to identify effects in organs that demonstrate effects in the chronic study. This abstract does not necessarily reflect U.S. EPA policy.

EFSA’s roadmap on NAMs and related case studies
George E. N. Kass, PhD, European Food Safety Authority (EFSA)
The European Food Safety Authority (EFSA) carries out risk assessments in relation to food and feed safety, and in the case of chemicals, provides health-based guidance values. These are mostly derived from in vivo animal toxicity data, using traditional endpoint measurements. However, EFSA is following closely and investing in the development and regulatory application of new methodologies and tools, the so-called New Approach Methodologies or NAM as part of its scientific strategy. The use of NAMs in regulatory chemical risk assessment has so far been very limited in EFSA. To overcome these challenges and generate opportunities for NAMs in regulatory chemical risk assessment, EFSA launched a contract to develop a roadmap for action on NAMs to reduce animal testing. The roadmap aims to define EFSA’s priorities for the incorporation of NAMs as well as to inform a multiannual strategy for increasing the use of NAMs in human health risk assessment to minimise the need for animal-based verification studies. Ultimately, it envisions that by 2027 the majority of EFSA’s requests for additional data will be based on NAMs. The presentation will provide an overview of the roadmap on NAMs and some of the activities currently ongoing in the context of its implementation.  

August 30, 2022 at 10:00-11:00am EDT / 14:00-15:00 CET

3D Molecular modelling meets toxicology: a useful tool to investigate the TD and TK of food related toxicants
Luca Dellafiora, PhD
The term “3D molecular modelling” refers to a group of computational methodologies relying on the 3D structures of biological (macro)molecules that study inter-molecular interactions from an atomistic and mechanistic standpoint. Such techniques have been widely used in medicinal chemistry and have succeeded to precisely study the molecular basis of low-molecular weight compounds’ activity, substantially supporting drug design during the last decades.
More recently, 3D molecular modelling techniques have been also successfully applied to investigate the toxicodynamics of food related toxicants like mycotoxins and other contaminants allowing either the precise understanding of their mechanisms of action or the prioritisation of molecules for more detailed experimental analysis. Moreover, those techniques have been usefully applied also to study the toxicokinetics of small molecules. In this context, we have applied 3D molecular modelling to study specific kinetic aspects of food-relevant chemicals ochratoxin A (OTA), alkenylbenzenes and perfluoralkyl substances (PFASs).
Our work focused on the transporter kinetics involved in the urinary excretion and reabsorption of members of the PFAS family and OTA by Organic Anion Transporter 4 (OAT4) and OAT1, respectively, and on the cytochrome P450 mediated biotransformation of OTA and alkenylbenzenes, with a focus on inter- and intra-species differences. Overall, our outcomes provided sound mechanistic basis supporting reported in vitro and in vivo data on toxicokinetics and may therefore also prove useful in the prediction of toxicokinetics for chemicals for which these data are lacking.

Measuring and modeling the distribution of test chemicals in in vitro toxicity assays
Nynke L. Kramer, PhD
In vitro toxicity assays lie at the heart of next generation risk assessment. A battery of in vitro assays provide concentration response relationships to extrapolate to bioequivalent doses in humans, a process referred to as quantitative in vitro-in vivo extrapolation (QIVIVE). The importance of in vivo toxicokinetic processes (absorption, distribution, metabolism and excretion, ADME) in driving toxicity is well acknowledged. That these processes also drive toxicity in in vitro assays and contribute to the variability and uncertainty of in vitro readouts is less well recognised. Generally, the nominal effect concentration, i.e., the concentration added to the exposure medium at the start of the exposure, is used to compare toxic potencies of test chemicals and assay sensitivity. However, chemicals differentially bind to serum constituents and well plate plastic, evaporate and accumulate in cells. Due to these kinetic processes, the concentration at the molecular target, which is ultimately responsible for the effect, is a fraction of the nominal concentration and differs per chemical and between assays. In this presentation, tools to measure and model this fraction are explained and the consequences of accounting for in vitro disposition kinetics for QIVIVE are discussed.

July 28, 2022 at 1:00-2:00pm EDT / 19:00-20:00 CET

Guidance for increasing confidence in Physiologically Based Kinetic models … are we ready for a regulatory change?
Alicia Paini, PhD, ERT, esqLABS GmbH
Physiologically based kinetic (PBK) models are routinely applied in several sectors, including academia and industry, as a means of integrating data on absorption, distribution, metabolism, and excretion (ADME) in humans and animals to predict whole-body biokinetic behavior. Use of these models has increased, especially in conjunction with emerging alternative methods to animal testing, now called new approach methodologies (NAMs). Experimental information derived from NAMs can be used as input for model parameterization and allows for increased confidence in models for chemicals that are lacking in vivo data for model calibration and evaluation. Despite significant advancements in good modelling practice (GMP) for model development and evaluation, there remains some reluctance among regulatory agencies to use such models during the risk assessment process. To fill this gap the Organisation for Economic Cooperation and Development (OECD) published a guidance document (GD) describing a scientific workflow for characterising and validating PBK models developed using NAMs data and with a lack of in vivo data for model evaluation. The GD provides an assessment framework for evaluating these models, with emphasis on the major uncertainties underlying model inputs and outputs. To help end-users develop and evaluate a PBK model for regulatory purposes, the document includes a template for documenting the model characteristics, and a checklist for evaluating the quality of a model. It was designed to facilitate the dialogue between model developers and risk assessors. The following presentation will highlight the principles, criteria and tools laid out in the OECD PBK model GD, with insight into the journey that lead to this document.

Implementing Good In Vitro Method Practices (GIVIMP) as a Quality Standard in a Laboratory
Amanda Ulrey, Institute for In Vitro Sciences, Inc. (IIVS)
Over the past several years science has put renewed energy into improving the reproducibility of data. There are many articles, strategies, and tools to help with this that are specific to the field of in vitro toxicology, such as systematic reviews of published work (and tools to help educate on how to perform them), publishing Good Cell Culture Practices (and GCCP 2.0), improvements made to the method validation process itself, and increased acceptance and use of in vitro methods within the Good Laboratory Practices (GLP) quality system. Another tool for improving the reproducibility of in vitro work is the implementation of the practices described in the OECD’s guidance document Good In Vitro Method Practices, or GIVIMP. The GIVIMP guideline is applicable to academic laboratories developing new alternative methodologies, established laboratories participating in validations and performing routine in vitro studies, and industry laboratories intending to submit in vitro data to regulatory agencies. These diverse types of institutions have different capabilities for developing and instituting quality systems that adhere to published recommendations while also suiting the unique operational environment of their facilities. This webinar will provide examples of and share a practical strategy for using the GIVIMP document to assess current laboratory processes and increase the reliability and robustness of work performed within a facility. GIVIMP has been used to prioritize areas of focus for building new and expanding existing good quality practices within the laboratory.

Wednesday, June 27, 10:00 AM – 11:00 AM EDT/16:00-17:00 CET

Registration is open!

This webinar discusses the impact of science on policymakers and provides advice on how to bring science to their attention. The webinar is dedicated to researchers and research organizations aiming to achieve policy impact. Attendees will hear from two EU and US speakers on the skills and best practices for researchers and the elements of institutional change necessary to bring scientists and policymakers into effective communication. Our third guest, Dr. Nataliia Bubalo of Ukraine, will then speak to her experience working to bridge the science/policy gap and promote the implementation of NAMs in Ukraine. Through this broad examination of experts’ experiences around the world, this webinar seeks to highlight knowledge areas and identify processes in which to invest.

The collected registration fees will be used for direct support of Ukraine via the organization People in Need.

A $10 USD registration fee is required to attend. If you would like to donate to People in Need beyond this amount, please follow this link.

Thank you for your support!

Moderators of the session: Helena Kandarova, ESTIV / Erin Hill, ASCCT

Speakers:
Kristie Sullivan, MPH, Physicians Committee for Responsible Medicine
Francois Busquet, PhD, Altertox
Nataliia Bubalo, PhD, National University of Food Technologies, Ukraine

Monday, May 16, 2022

11:00 AM -12:00 PM EDT / 17:00-18:00 CEST

Registration is open!

A novel prediction model to evaluate genotoxicity based on biomarker genes in human HepaRGTM cells
Presented by Anouck Thienpont, PhD student, Laboratory of In Vitro Toxicology and Dermato-Cosmetology
Transcriptomics-based biomarker discovery is a promising new approach methodology (NAM) to identify molecular events underlying the genotoxic mode of action of chemicals. Previously, we developed the GENOMARK biomarker, consisting of 84 genes selected based on whole genomics DNA microarray profiles of 24 (non-)genotoxic reference chemicals covering different modes of action in metabolically competent human HepaRG™ cells. In the present study, new prediction models for genotoxicity were developed based on an extended reference dataset of 38 chemicals including existing as well as newly generated gene expression data. Both unsupervised and supervised machine learning algorithms were applied to the extended dataset, but as unsupervised machine learning was not able to clearly distinguish both groups, the performance of two supervised machine learning algorithms, i.e. support vector machine (SVM) and random forest (RF), was evaluated. More specifically, the predictive accuracy was compared, the sensitivity to outliers for one or more biomarker genes was assessed and the prediction performance for 10 misleading positive chemicals exposed at their IC10 concentration was determined. Furthermore, the applicability of both prediction models on a public gene expression dataset, generated with RNA-sequencing, was investigated. Overall, the RF and SVM models showed to be complementary in their classification of chemicals for genotoxicity. To facilitate data analysis, an online application was developed, combining the outcomes of both prediction models. Furthermore, this research demonstrates that the combination of gene expression data with supervised machine learning algorithms can contribute to the ongoing paradigm shift towards a more human-relevant in vitro genotoxicity testing strategy.

A novel fluidic microphysiological system for 3D cancer tissue culture towards a better understanding of human cancer progression
Presented by Silvia Scaglione, React4life, ESTIV corporate member
The medical research focused to better understand human cancer disease and the preclinical assessment of novel drug therapies are currently carried out through traditional too simplistic 2D cell culture, and in vivo animal models, whose cost, time, and poor predictivity remain important challenges.
A novel Multi-In Vitro Organ (MIVO) organ on a chip platform has been recently developed to culture human tumor models under physiological culture conditions. Biologically relevant cancer samples were cultured within the MIVO chamber under blood capillary fluid flows that (i) feed the 3D tumor tissue, (ii) resemble cancer cells migration and intravasation through a permeable barrier resembling the vascular barrier, and (iii) supporting their migration towards metastatic target tissues. The same experimental approach was adopted to test the efficacy of an anticancer drug that was placed in the fluidic circuit, mimicking the systemic drug route. The regression of the ovarian tumor was observed and measured within the MIVO device: results were in line with the in vivo data, with a significant time reduction (75%). Similarly, a human 3D neuroblastoma model with proper immunophenotype was optimized to develop a complex tumor/immune cell coculture within the MIVO as paradigm of an immune-oncology screening platform. Immune cells have been introduced within the capillary fluid flow circulation of the MIVO and their migration and infiltration towards the 3D tumor model was analyzed. Flow cytometry analysis highlighted a tumor mediated immune cells extravasation and infiltration, with an initial reduced tumor cell viability.
In conclusion, we generated a functional and relevant human cancer model, through the adoption of MIVO device, that can be efficiently employed as an drugs screening platform, both for pharmacological treatments and for cell-based therapies, and for basic research purposes.

Evaluation of in vitro New Approach Methodologies for Developmental Neurotoxicity
Presented by Kelly Carstens, PhD, US EPA ORISE
Current developmental neurotoxicity (DNT) hazard assessment relies on in vivo testing that is resource intensive and lacks information on key cellular processes. To address these limitations, DNT new approach methodologies (NAMs) are being evaluated for their utility to inform DNT hazard, including: functional microelectrode array network formation assay (NFA) to evaluate neuronal network formation; and high-content imaging to evaluate proliferation, apoptosis, neurite outgrowth, and synaptogenesis. This work applies computational approaches to address three related hypotheses: (1) a broad screening battery will provide a sensitive marker of potential DNT bioactivity; (2) evaluating selective bioactivity may provide a more specific indicator of the functional processes underlying DNT; (3) some subset of the DNT NAM endpoints may optimally classify DNT in vivo reference chemicals. The dataset contained 57 assay endpoints, with a union set of 92 screened chemicals, including 53 in vivo DNT reference positives and 13 putative negatives. K-means clustering of selectivity revealed five chemical clusters with distinct DNT-relevant activity and identified DNT reference compounds with 66% sensitivity and 92% specificity, capturing 18 false negatives. We used a supervised machine learning approach to identify the most informative endpoints in classifying a DNT reference chemical. The five most important features included three cytotoxicity endpoints, network formation, and neurite outgrowth endpoints. This work highlights current obstacles in DNT NAM data interpretation, including a limited set of reference negatives and the question of whether cytotoxicity should be considered off-target or is relevant to deriving key DNT-related points-of-departure. Together, these data emphasize the importance of an integrated analysis that combines computational approaches, a broad chemical set, and a diverse suite of assays to demonstrate the fit-for-purpose utility of DNT NAMs for identification, hazard characterization, and prioritization of chemicals. This abstract does not reflect EPA policy.

A 3D alveolar in vitro model for the prediction of chemical respiratory sensitizers and irritants
Presented by Sabina Burla, Luxembourg Institute of Science and Technology (LIST)
The increasing prevalence of chemical respiratory allergic diseases resulting in high morbidity and mortality deems necessary a precise classification and labelling of chemicals inducing respiratory sensitization, to ensure the hazard is communicated and allow the safe handling and use. The immunological mechanisms underlying the development of respiratory sensitization are not fully understood, and the available new approach methodologies (NAMs) for identification of skin sensitizers fail to categorize chemicals as respiratory or skin sensitizers. Therefore, physiologically relevant test systems for the respiratory tract are essential to anticipate the sensitizing potential of chemicals.
The aim of the present study was to evaluate the performance of a 3D alveolar-capillary barrier designed for the prediction of respiratory sensitizing chemicals. The model is built using human cell lines: alveolar type II epithelial (A549), endothelial (EA.hy926) and monocyte (THP-1). The cellular arrangement within the test system favors the development of a tissue-like microenvironment by cell-to-cell direct communication and indirectly through the secreted messenger molecules. Additionally, it facilitates the exposure of respiratory sensitizers in a more realistic approach, at the air-liquid-interface (ALI).
A panel of test items including known respiratory sensitizing chemicals and irritants was used for exposures to assess the capacity of the test system. Cell viability was measured in the resazurin assay 24h post-exposure at ALI, and the dose-response curves were modelled for the apical, basolateral compartments, and the complete test system. The test system was subsequently exposed to a test item dose leading to a cell viability of at least 75% and the expression of CD54, CD86 and TSLPr cell surface markers was measured by flow cytometry.
Results show that the test system has the potential to distinguish respiratory sensitizers from irritants. In addition, tested pro-haptens were correctly identified as respiratory sensitizers, chemicals reported as false negatives by other NAMs for respiratory sensitization evaluation.

Thursday, March 17 2022, 9:00-10:30 am EST / 15:00-16:30 CET

Full-thickness skin-on-a-chip model for in vitro drug testing and disease modelling
Presented by Patrícia Zoio, PhD candidate, Institute of Chemical and Biological Technology, NOVA University of Lisbon

Reconstructed human skin models are a valuable tool for drug discovery, disease modeling, and basic research. However, currently available skin models do not fully reproduce the structure and function of the native human skin, mainly due to their use of animal-derived collagen and their lack of a dynamic flow system. Here, we present a full-thickness skin-on-a-chip (SoC) system that reproduces key aspects of the in vivo cellular microenvironment. This approach combines the production of a fibroblast-derived matrix (FDM) with the use of an inert porous scaffold for the long-term, stable cultivation of a human skin model. The use of a scaffold resulted in a structure with mechanical stability due to its noncontracting nature. The coculture of primary human keratinocytes resulted in a terminally differentiated skin equivalent that could maintain its architecture and homeostasis for up to 50 days. The developed SoC resulted in a differentiated epidermal compartment with increased thickness and barrier function than tissues cultured under static conditions. Integrated electrodes were used to measure transepithelial electrical resistance (TEER) during tissue culture, quantitatively evaluating tissue barrier function. After 14 days of culture, the barrier tissue was challenged with a benchmark irritant and its impact was evaluated on-chip through TEER measurements. In the future, this innovative low-cost SoC with integrated electrodes could provide a new in vitro tissue system compatible with long-term studies to study skin diseases and evaluate the safety and efficacy of novel drugs.

Developing and investigating a new in vitrohepato-pulmonary coculture model for the toxicological study of inhaled xenobiotics

Presented by Sabrina Madiedo-Podvršan, PhD candidate, Université de Technologie de Compiègne (UTC)

The respiratory system is exposed daily to a wide range of airborne xenobiotics. Associated toxicity mechanisms are complex and involve local and systemic responses which aggravate, stabilize, or protect our body from adverse effects. Because of this complexity, animal models are considered prime study models. However, in the European context of animal experimentation reduction, we developed a new alternative method to consider dynamic interactions between the lungs and a detoxicating organ such as the liver. According to literature, both organs reportedly interact under stress, therefore our goal is to consider any eventual toxicity modulating crosstalk behaviors
following exposure. Two kinds of Lung/Liver (LuLi) models have been developed: a developmental model which allowed for the technical setup of the coculture platform and a physiological-like model which better approximates a human vivo situation. Coculture models were characterized using 72-hour acetaminophen (APAP) xenobiotic exposures. Morphology, viability, and functionality properties of both compartments were monitored following exposure giving us an insight into the passage and circulation of the model substance throughout the device. Results show that while APAP has adverse effects on both compartments, toxicity is decreased in a coculture setting. Thus, the developed LuLi model displays a relevantly active and functional inter-organ communication between both compartments. Further characterization of the developed lung-liver-on-a-chip is
ongoing but our results are promising and could open the way to a new physiologically relevant way of studying inhalation toxicology.

3D Human airway epithelial models to study SARS-CoV-2 pathogenesis and to discover antivirals
Presented by Samuel Constant, PhD, CEO, Epithelix
The recent outbreak of SARS-CoV-2 (COVID-19) is a major threat to human beings. The respiratory system is the main entry portal of SARS-CoV-2 which infects initially and principally the airway epithelia; then it gradually propagate to other human organs, causing symptoms such as fever, dry cough, fatigue, diarrhoea, conjunctivitis, pneumonia, respiratory failure, loss of taste, etc… To fight against SARS-CoV-2, confinement is necessary but not sufficient. Vaccination is certainly a priority, but new anti-viral drugs are also indispensable. Since the first step of SARS-CoV-2 infection is taking place in airway epithelial cells, it is logic to use 3D airway epithelial model as drug testing platform. Epithelix has developed and is offering standardized air-liquid interface 3D human airway epithelial cultures from nasal or bronchial (MucilAir™) and small-airway (SmallAir™) origins. These epithelial models closely mimic the morphology and function of the native tissues: such as cilia formation and beating, mucus production and secretion, mucociliary clearance, and secretion of antiviral molecules. These models have been successfully used for the development of antivirals against influenza, rhinoviruses, respiratory syncytial virus, amongst others.
This talk will highlight how these reconstituted human airway epithelial models can be used to characterize viral infection kinetics, tissue-level tropism and transcriptional immune signatures induced by SARS-CoV-2. Relevance of these models for the preclinical evaluation of antiviral candidates will also be addressed in the context of repositioning of marketed drugs or evaluation of novel therapies and combinations delivered systematically or through aerosol therapy.

Thursday, February 24 2022
9:30-10:30 AM EST / 15:30 -16:30 CET

Presenter 1: Barbara Jozef, PhD, EAWAG

Title: Nutritional requirements of fish cell lines: development of a serum-free culture medium for routine culturing of the RTgill-W1 cell line

Abstract:

Efforts to replace fish for regulatory testing have resulted in the development of strategies and assay procedures based on permanent fish cell lines, especially of rainbow trout (Oncorhynchus mykiss). The rainbow trout gill cell line, RTgill-W1, is at the core of an acute cytotoxicity assay to predict fish acute toxicity to chemical exposure. It is the first of its kind to be approved by international standardization organizations, namely the Organisation for Economic Co-operation and Development (OECD) and the International Organization for Standardization (ISO). However, though the assay as such is performed in a simple exposure medium without the addition of animal components, RTgill-W1 cells are grown routinely in Leibovitz’s L-15 medium (L-15) supplemented with 5–10% fetal bovine serum (FBS). FBS is a very complex, ill-defined mixture, which is additionally derived by a highly unethical method. We therefore have set out to develop a serum-free formulation capable of supporting RTgill-W1 cell proliferation through a systematic, bottom-up medium design.

The novel medium formulation enables RTgill-W1 cells to proliferate in the absence of serum. Adaptation of RTgill-W1 clones to this new serum-free medium, including attachment, passaging, and freeze/thawing, is well underway – we may not be far from having the first fully defined culture medium for at least one type of fish cell.

Presenter 2: Aline Chary, PhD, LIST

Title: Using FBS-free media in in vitro cell cultures: A case study in transitioning and characterizing A549 cells

Abstract:

Replacing the use of fetal bovine serum (FBS) in cell culture media bolsters the reproducibility of in vitro research and overcomes the ethical and legal challenges associated with its use. Increasingly, scientists are focusing on replacing the use of FBS as a supplement in cell culture media with animal-component-free media.

Using A549 cells—an immortalized human epithelial alveolar-like cell line commonly used in respiratory research—as a case study, we demonstrate the process of transitioning cells cultured in medium containing FBS to commercially-available media without FBS. To determine whether the transition was successful, cellular morphology and functionality were assessed by imaging (scanning electron microscopy); calculating cell doubling time, cytokine release (Bio-Plex), and cell viability (Alamar blue assay); monitoring the expression of relevant genes; and determining surfactant production (surfactant droplet test). Our results show that, while success varies based on the transition process and type of media, animal-derived components can be replaced in the culture of A549 cells. Because FBS-free media can replicate the phenotype of A549 cells similar to that observed in FBS-supplemented medium or a phenotype more similar to normal alveolar epithelial cells, the medium should be chosen based on the objective of the study. This case study can be used as a guide to transition other cell types to FBS-free media.

Friday, January 21, 2022
10:00-11:30 EST / 4:00-5:30 CET

Recording is available at: https://youtu.be/PPzRdm4I0gU

Presenters: 
Stefan Pfuhler, Procter and Gamble
Kerstin Reisinger, Henkel AG & Co. KGaA

The in vitro genotoxicity test battery has a high sensitivity for prediction of in vivo genotoxic/carcinogenic agents but tends to over-predict the genotoxicity hazard, resulting in misleading positive results. To address this, the Cosmetics Europe Genotoxicity Task Force has established two in vitro skin genotoxicity models as follow up assays to the in vitro test battery for substances with dermal exposure: the reconstructed skin (RS) Comet assay and the RS micronucleus (RSMN) test. Here, we report on the completed validation of these assays. Both assays exhibited good sensitivity and specificity: 77% and 88% for 3D Skin Comet (32 compounds) and 80% and 87% for the RSMN (47 compounds). A combination of these assays enables detection of DNA damage leading to all 3 types of genotoxic damage (mutation, clastogenicity and aneugenicity). By applying an endpoint-triggered strategy, the sensitivity increased to 89%. The high predictivity for the expected in vivo outcome observed for these higher tier in vitro assays supports their use as follow-up tests to the standard test battery. For topically applied chemicals, the RSMN assay is recommended for in vitro positive chemicals that showed a clastogenic or aneugenic MoA; whereas, substances triggering gene mutation should be followed-up with a RS Comet assay. This tiered strategy shows great promise as an in vitro-only approach for genotoxicity testing of dermally exposed substances and both assays were recently accepted into the OECD guideline development program.

This popular award winners series highlights the science of speakers who have been recognized by ASCCT and ESTIV in 2021.

Thursday, December 16, 2021
10:00-11:30 EST / 4:00-5:30 CET

The recording is available here: https://www.youtube.com/watch?v=UQzy50rUEgY&t=1s

Presenter 1: Agnes Karmaus, Integrated Laboratory Systems/NICEATM
Title: Gaining Context for High-Throughput In Vitro Data: Annotation Approaches and Visualization Tools

Availability of in vitro high-throughput screening (HTS) assay data is facilitating the development of computational approaches for chemical hazard assessment. Linking HTS data to regulatory endpoints remains a challenge and requires detailed information about assays as well as an understanding of biological context. For example, data from the U.S. Environmental Protection Agency’s ToxCast HTS program are annotated by technology platform, assay design, and assay/gene target information, yet it remains a challenge to provide toxicological context for potential regulatory applications. Here we present a mapping approach for HTS assay endpoints that moves beyond technology-based assay annotations. Our mapping provides a robust assay grouping schema applicable beyond HTS datasets in a toxicological endpoint-based framework. This expert-led curation and annotation is available in the Integrated Chemical Environment (ICE). Annotations map assays to regulatory toxicological endpoints of interest through structured vocabularies allowing data to be searched, grouped, and visualized by regulatory endpoint. The annotations increase accessibility for those unfamiliar with individual assays by providing context for in vitro assays (and in vivo data or in silico predictions in ICE) to facilitate identification of data gaps, insight into mechanistic plausibility, and investigation into regulatory-relevant endpoints. Finally, we highlight that while single assay results are generally insufficient for regulatory application, this approach helps integrate results from multiple assays and provides data visualization to aid review of a chemical’s potential activity for selected regulatory endpoints. This project was funded with federal funds from the NIEHS, NIH under Contract No. HHSN273201500010C.

Presenter 2: Saskia Galanjuk, IUF-Duesseldorf
Title: The human induced pluripotent stem cell (hiPS) test as an alternative method for developmental toxicity testing

The evaluation of substances for their potency to induce embryotoxicity is controlled by safety regulations. Test guidelines for reproductive and developmental toxicity are relying mainly on animal studies which make up the majority of animal usage in regulatory toxicology. Therefore, there is an urgent need for alternative in vitro methods to follow the 3R principles. To improve human safety, cell models based on human cells are of great interest to overcome species differences. Human induced pluripotent stem cells (hiPSCs) are an ideal cell source as they largely recapitulate embryonic stem cells without bearing ethical concerns and they are able to differentiate into most cell types of the human body. Here, we set up and characterized an FBS-free hiPSC-based in vitro test method, called the human induced pluripotent stem cell test (hiPS Test), to evaluate the embryotoxic potential of substances. After 10 days in culture, hiPSCs develop into beating cardiomyocytes. As terminal endpoint evaluations, cell viability, qPCR analyses as well as contraction frequency and area of beating cardiomyocytes by video analyses are measured.

Presenter: Oliver Wehmeier, acCELLerate GmbH

November 30, 2021
10:00-11:00 AM EST / 16:00-17:00 CET

The recording is available here: https://www.youtube.com/watch?v=YxFwIFEVjgI

Human or animal derived cell lines are major cornerstones for the development of alternative methods. An increasing number of animal tests can now be replaced by cell-based assay. The various validated methods provide a good predictivity and high precision, but the reliability of the assays very much depends on the quality of cell culture. Although considered as in vitro, cells are still living organisms.

There are many determinants which modulate the proper function of cells and have an impact on the outcome of an assay. To know what these determinants are and to understand how they can be controlled and standardized is the holy grail of cell culture. Ways to prevent microbial contamination, the importance of regular mycoplasma testing as well as the problem of cross contamination among cell lines will be discussed. Also, variances which are caused from the cells and the cultivation process itself will be addressed. The guidance for a Good Cell Culture Practice as the essential basis for running reliable and precise cell-based assays will be introduced.

Thursday, October 21, 2021
11:00 AM ET – 12:00 PM ET / 17:00 – 18:00 CET

Presenters:
Costanza Rovida, PhD, CAAT-Europe/Konstanz University
Jean Knight, White Rabbit Beauty

The recording is available here: https://www.youtube.com/watch?v=d_lQJ94jGdU

EU REACH Regulation, EU Cosmetic Regulation, and new animal tests on cosmetic ingredients

Presenters: Costanza Rovida, PhD, CAAT-Europe/Konstanz University and Jean Knight, White Rabbit Beauty

EU Regulations are legislative acts that are immediately implemented in all 27 member states of the European Union. REACH is the most important regulation in the chemical area, requiring  all manufacturers and importers of chemicals in quantities above 1 ton/year to register the substances. The registration includes a detailed risk assessment, often based on new animal testing. Cosmetic ingredients are chemical substances that need full REACH registration. In addition to that, cosmetic ingredients are governed by the EU’s Cosmetic Regulation, which is focused on the protection of consumers when using cosmetics. Since 2009 for some endpoints and 2013 as a general rule, the Cosmetic Regulation has banned new in vivo tests of both finished cosmetics and cosmetic ingredients. This important ban has caused consumers to believe that EU cosmetics are not tested on animals. In a recent study, we demonstrated that this is not fully true, as many cosmetic ingredients are being tested in vivo for REACH purposes (https://doi.org/10.14573/altex.2104221).

During the presentation we will describe both regulations and we will guide participants through the database of the European Chemical Agency (ECHA) where all the studies performed for registering chemical substances are stored. We will also explain the conflict between the two regulations regarding the possibility to test cosmetic ingredients on animals, by presenting the results of our analysis. Among the in new in vivo tests performed on cosmetic ingredients, some were necessary for REACH compliance, but others should have been avoided. The future is not bright with many more new tests asked by the authority to the registrants.

Presenter/s:
Meike van der Zande, PhD, Wageningen Food Safety Research
Monica Piergiovanni, PhD, European Commission Joint Research Centre

The recording is available here: https://www.youtube.com/watch?v=r-lXYx6Y5mE

Organ-on-Chip models for bioavailability, biotransformation, and (sub)chronic toxicity studies

Presented by Meike vander Zande

Organ-on-Chip (OoC) systems are promising tools for improvement of current in vitro models. Microfluidic flow conditions allow for accurate control of the extracellular chemical and physical microenvironment in which cells are grown, thus better recapitulating the in vivo tissue environment. As such, these models may facilitate the replacement of animal models.

During this presentation, two types of organ-on-chip models will be discussed, namely; intestinal and liver OoC systems. The intestinal systems vary in complexity ranging from a model based on Caco-2 epithelial cells to a tri-culture model composed of Caco-2, HT29-MTX (Goblet) and hMVEC (endothelial) cells. The liver model is comprised of HepaRG cells.

The design and characterization of the models will be discussed, focusing on critical parameters that should be evaluated. Furthermore, the models will be discussed in relation to their performance for bioavailability, biotransformation, and (sub)chronic toxicity studies, showing their potential for future application in the fields of pharmacology and toxicology.

The many roles of standards for Organ on Chip

Presented by Monica Piergiovanni

Organ-on-Chip (OoC) refers to a group of innovative technologies able to maintain and monitor living engineered human or animal tissues in a controlled microenvironment. The purpose of OoCs is to mimic specific aspects of organ physiology and function, including interactions between different organs in the body.

Standards are commonly seen as a means to bring innovation to the market, strengthening the industry and contributing to the Single Market strategy that we have in place in Europe. Standard methods are also great tools to provide a basis for regulatory acceptance, facilitating the use of new technologies, as is the case for alternative to animal methods.

There is a clear consensus in the OoC community on the need for standardisation to advance the field, with several initiatives now in progress worldwide. Standardisation can play a key role in ensuring proper characterisation of individual devices, benchmarking against appropriate reference elements and aiding efficient communication among stakeholders. This was also the subject of the join workshop of the European Commission Joint Research Centre, (EC-JRC) together with the European Standardization authorities (CEN/CENELEC). The workshop had the goal to bridge the research and developers community with the standardisers and regulators and build a list of areas where standards would contribute to wider acceptance and use of OoC. Standards can tackle multiple aspects of the OoC, from assessment of technical performance to material characterisation, from reference compound to cell sources, from reporting to quality control.

Tuesday, June 23, 2021
10:00 – 11:30 EST / 16:00 – 17:30 CET

The recording is available here: https://www.youtube.com/watch?v=phZY4WnNp2o

Presenters:
Catherine Verfaillie, Leuven University
Anne Marie Vinggard, National Food Institute, Technical University of Denmark

Abstracts: “Multicellular 3D liver models based on hiPSC-derived liver cells“
Presented by Catherine Verfaillie

To address how toxic insults affect hepatocytes as well as non-parenchymal cells, murine or rat models are often used. However, interspecies differences between humans and rodents do not always allow extrapolating findings in animal models to the human patient. Therefore, human hepatoma cell lines with or without stellate cell, monocyte, and/or endothelial cell lines can be used. In addition, and more relevant, primary human liver cells can be used. However, It has long been established that when cultured on stiff culture plates, primary human hepatocytes (PHHs) very quickly lose functionality. This is also the case for hepatic stellate cells (HSCs)  that become quickly activated, and liver sinusoidal endothelial cells (LSECs) that very quickly lose their typical sinusoidal endothelial characteristics. Especially for hepatocytes, numerous studies have demonstrated that this can at least in part be prevented when cells are cultured for instance in sandwich cultures, in spheroids or embedded in scaffolds. Although the latter best mimic human liver, the scarcity of human liver cells and high inter-donor variation  represent important drawbacks. With the advent of pluripotent stem cells (PSCs), it is now possible to create PSC-derived hepatocyte-, HSC-, EC- and macrophage (Mφ)-like cells.  As PSCs are an inexhaustible cell population, this approach overcomes the issues related to low cell numbers and inter-donor variability of primary samples. To create more complete liver models, co-cultures of PSC-hepatocyte-like cell (HLC) progeny with mesenchymal cells and human umbilical vein endothelial cells (HUVECs) have been created, and more recent studies created liver models by co-differentiation of PSCs to hepatocyte- and NPC-like cells directly in spheroids. An alternative is the assembly of pre-differentiated PSC-HLCs and -NPC-like cells into co-cultures. During this presentation, data will be presented from studies exploiting the latter concept to create in vitro liver models that are stable for several weeks, and can be used to test drug metabolisation, drug toxicity or induction of liver inflammation and fibrosis.

“Novel human pluripotent stem cell-based assays to predict developmental toxicity“
Presented by Anne Marie Vinggard

As women are exposed to a plethora of chemicals during pregnancy, it is important to evaluate their safety for the developing embryo. For this, human based models are needed for enabling reliable and large scale risk assessment1. Human induced pluripotent stem cells (hiPSC) are an excellent tool to model embryonic development as they can form embryoid bodies (EBs) that mirror the early embryo in many structural and functional aspects, and can undergo differentiation programs that reflect molecular mechanisms during embryonic development2. Here, we present our recently developed assays for predicting developmental toxicity using hiPSC.

We used three different hiPSC lines and differentiated the cells through EBs into cardiomyocytes. The cells were exposed to chemicals for the last six days of differentiation and the beating EBs analyzed on the last day4. In order to characterize our system and to study potential molecular mechanisms-of-action, we also investigated global and targeted gene expression levels and protein cardiac markers.  We were able to show that our system mimics the first three weeks of embryonic development by analyzing markers for respective developmental stages3. Furthermore, the assay was able to detect the strong developmental toxicant thalidomide as positive while the neurodevelopmental toxicant valproic acid was negative. Interestingly, we also identified the fungicide epoxiconazole as a potentially hazardous chemical4 and studied the underlying mechanism-of-action. Moreover, we have created a human NKX2.5 reporter stem cell line for developmental toxicity testing with a greater sensitivity and a higher throughput5. In conclusion, we have developed two assays based on hiPSC that can predict developmental toxicity and that reflect key aspects of early embryonic development.

April 21, 2021
10:00 – 11:00 EST / 16:00 – 17:00 CET

Thursday, February 25th, 2021
10:00AM – 11:30AM EST

• Presenters:
• Martin Scholze, Centre for Pollution Research and Policy, Brunel University London, UK
• Ans Punt, Wageningen Food Safety Research, The Netherlands

The recording is available here: https://www.youtube.com/watch?v=TN-iMQxwWh0

Abstracts

Martin Scholze, Centre for Pollution Research and Policy, Brunel University London, UK
Quantitative in Vitro to in Vivo Extrapolation (QIVIVE) for Predicting Reduced Anogenital Distance produced by Anti-Androgenic Pesticides in a Rodent Model for Male Reproductive Disorders

Many pesticides can antagonize the androgen receptor(AR) or inhibit androgen synthesis in vitro but their potential to cause reproductive toxicity related to disruption of androgen action during fetal life is difficult to predict. Currently, no approaches for using in vitro data to anticipate such in vivo effects exist, and prioritization schemes that limit unnecessary animal testing are urgently needed. Here I will present a quantitative in vitro to in vivo extrapolation (QIVIVE) approach for predicting in vivo anti-androgenicity arising from gestational exposures and manifesting as a shortened anogenital distance (AGD) in male rats. It is built on a physiologically based pharmacokinetic (PBK) model to simulate concentrations of chemicals in the fetus resulting from maternal dosing. The QIVIVE approach was evaluated by using several well-studied model compounds and then applied to various current-use pesticides, all with in vitro evidence for anti-androgenicity but missing in vivo data. Here the QIVIVE model newly identified fludioxonil, cyprodinil, and dimethomorph as in vivo anti-androgens. With the examples investigated, this approach shows great promise for predicting in vivo anti-androgenicity (i.e., AGD shortening) for chemicals with in vitro activity and for minimizing unnecessary animal testing.

Ans Punt, Wageningen Food Safety Research, The Netherlands
A web-based toolbox to support quantitative in vitro-to-in vivo extrapolations (QIVIVE)

An important aspect within next generation non-animal toxicity testing strategies is the extrapolation of in vitro effect concentrations into (human) dose−response or potency information, also called quantitative in vitro-to-in vivo extrapolation (QIVIVE). Insights in dose-dependent plasma and tissue concentrations of a chemical are required to infer the dose levels that would be needed to reach the in vitro effective concentrations in the body. Physiologically based kinetic (PBK) modelling plays a crucial role in generating such insights. To support PBK-model development we have developed a web-based toolbox (www.qivivetools.wur.nl) that contains generic physiologically based-kinetic (PBK) models for rats and humans, including underlying calculation tools to predict plasma protein binding and tissue:plasma distribution. The PBK models within the toolbox allow to make first estimations of internal plasma and tissue concentrations of chemicals, based on the LogP and pKa of the chemical and values for intrinsic hepatic clearance and intestinal uptake. As case study, the toolbox was used to predict oral equivalent doses of in vitro ToxCast bioactivity data for the food additives methylparaben, propyl gallate, octyl gallate and dodecyl gallate. These oral equivalent doses were subsequently compared with human exposure estimates. The goal of the presentation is to provide a background on PBK model development and QIVIVE, making interactively use of the web-based toolbox.

Thursday, January 14 2021                                                                                                                                                     10:00-11:15  AM US ET// 4:00 PM – 5:15 PM CET

The recording is available here: https://www.youtube.com/watch?v=pulEcFwaOPw

Awards

The Ed Carney Predictive Toxicology Award recognizes the first author of an outstanding poster or platform presentation that will advance predictive toxicology, and is named after Dr. Ed Carney, a member and supporter of the ASCCT and a leader in the predictive toxicology field. The 2020 winner was Eva C.M. Vitucci from University of North Carolina, Chapel Hill to for her presentation “Identifying the Molecular Mechanisms of Air Pollution-Induced Cardiovascular Disease”.

The Ray Tice Tox21 Student Award is presented to recognize the best student presentation during the meeting and was established with a generous donation from Dr. Ray Tice, a leader in implementing and setting the stage for Toxicity Testing in the 21st Century. The 2020 winner was Ms. Sherri Bloch from Université de Montréal for her presentation “Using in Vitro Data to Derive Acceptable Exposure Levels: A Case Study on PBDE Developmental Neurotoxicity”.

Abstracts

Using in vitro data to derive acceptable exposure levels: Case studies on PBDE
developmental neurotoxicity and DDE obesogenicity
Presented by: Sherri Bloch

Current acceptable exposure levels are mostly based on animal models, which are costly,
time-consuming, and may poorly predict adverse outcomes in humans. There is a need for
alternative testing methods that are faster, cheaper, and provide human-relevant information.
We aimed to evaluate a method using human in vitro data and biological modeling to calculate acceptable exposure levels through two case studies: One on PBDE developmental neurotoxicity and the other on p,p’-DDE obesogenicity due to early life exposure.
To accomplish our objectives, in vitro studies using human cells were compiled to select points of departure (POD) in the form of nominal medium concentrations. These were translated into cellular levels (target organ POD), and then converted into acceptable daily intakes and plasma concentrations in pregnant women using pharmacokinetic models of gestation/lactation and applying uncertainty factors. The final step of our methodology was the comparison of the derived chemical levels in maternal plasma to levels measured in epidemiological studies reporting associations between prenatal exposure and the adverse endpoints in children.
Estimated acceptable exposure levels and matching plasma levels showed concurrence with those measured in epidemiological studies, implying this approach may be viable. However, uncertainty factors need further investigation prior to application in risk assessment.

Identifying the Molecular Mechanisms of Air Pollution – Induced Cardiovascular Disease

Presented by: Eva Vitucci

An estimated 3.5-million people die annually from air pollution-induced cardiovascular disease (API-CVD). API-thrombosis (API-T) is a main contributor of these mortalities; however, the molecular mechanisms driving API-T are unclear. To identify these mechanisms we developed an in vitro model that represents the interface of the respiratory and cardiovascular system, the alveolar capillary region (ACR). This organotypic model includes human alveolar-like epithelial cells (H441), human lung fibroblasts, and human lung microvascular endothelial cells (HULEC). We hypothesized that air pollutant exposure of the H441 cells would initiate the onset of a pro-thrombotic state in the HULEC. To test this, we exposed H441 cells to the ubiquitous air pollutant, diesel exhaust particulates (DEP), and investigated the effect of this trans-alveolar exposure (TA-DEP) on the underlying HULEC. TA-DEP exposure induced expression of anti-oxidants such as heme oxygenase 1 and NAD(P)H dehydrogenase [quinone] 1, increased nuclear factor erythoid-2 related factor (NRF2) protein, and reduced glutathione redox potential in the HULEC. Concurrently, decreased expression of the endothelial fibrinolytic and anti-coagulant genes, tissue-type plasminogen activator, plasminogen activator urokinase, and thrombomodulin, and increased expression of the procoagulant gene, coagulation factor III occurred in the HULEC. These data suggest that TA-DEP exposure induces redox dysfunction and an endothelial pro-thrombotic profile in the ACR. We conclude that redox dysfunction and pro-thrombotic activation in the capillary beds of the ACR may be critical initiation steps of API-T. Data from this model can help develop intervention strategies against API-T and can encourage the development of organotypic models resulting in a reduction of animal testing. This does not reflect EPA policy.

Speakers

Thursday, December 17th, 2020
11:00-12:30  AM US ET// 5:00 PM – 6:15 PM CET

In vitro methodology for medical device material thrombogenicity assessments

Presenter: Michael F. Wolf, PhD, Medtronic

In vivo vascular implant models are currently used to assess implant material thrombogenicity for medical devices. We report an in vitro thrombogenicity assessment methodology where test materials alongside positive and negative controls were exposed to fresh human blood, using gentle-agitation test tube and pulsatile-flow closed-loop models. We report that: thrombogenicity was strongly dependent on several experimental variables, and in vitro to in vivo case comparisons showed consistency in thrombogenicity rankings on a range of materials. In vitro methods using fresh human blood are scientifically sound and cost effective compared to in vivo methods for screening thrombogenicity.

In Vitro Irritation Testing for Medical Devices: A Progress Update

Presenter: Kelly P. Coleman, PhD, DABT, ERT, Medtronic

Assessment of skin irritation is an essential step in the biocompatibility evaluation of medical devices. Two international validation studies were conducted to evaluate reconstructed human epidermis (RhE) models as alternatives to the rabbit skin irritation test. First, extracts of four irritant polymers and three non-irritant controls were used with two types of RhE tissues. Both tissue types identified virtually all the irritant polymer samples, indicating that RhE tissue models can detect the presence of strong irritants at low levels in dilute medical device polymer extracts. The second study used two other types of RhE tissues and produced similar results. It was concluded that RhE models are suitable replacements for the rabbit skin irritation test to support the biocompatibility evaluation of medical devices, and in 2020 a new ISO 10993-23 standard was finalized which states that the use of RhE tissues is preferable to rabbits.

Register here: https://register.gotowebinar.com/register/9037618754836657166

Monday, November 30 th, 2020
10:00-11:00  AM US ET// 16:00-17:00 CET

Presenter: Adam Lillicrap, Norwegian Institute for Water Research 

Reduction of animal testing, wherever possible, is required by legislations such as the EU Directive 2010/63/EU. Fish Embryo Toxicity (FET- OECD TG236) testing has been proposed to be an alternative to the juvenile acute fish toxicity test (AFT- OECD 203). However, FET data are not yet accepted as a replacement to the AFT test for certain regulatory purposes such as REACH. The European Chemicals Agency (ECHA) recommended that a weight-of-evidence (WoE) approach was needed for FET data before it could possibly be used in place of AFT data. Therefore, a Bayesian network (BN) model has been developed to incorporate multiple lines of evidence, in combination with FET data, to predict AFT. Bayesian networks are increasingly being used in ecological risk assessment because they can integrate large amounts of data and other information sources to produce discrete probability distributions, and predict the probability of specified states.

The objectives of this study were:

1) To develop and evaluate a BN model for predicting toxicity of substances to juvenile fish from embryo toxicity data in combination with other relevant information;

2) To apply the BN model in a WoE approach which can support replacing juvenile fish toxicity testing with embryo toxicity testing.

The BN model correctly predicted the AFT toxicity level for 14 substances, and gave lower toxicity for 6 substances. For the 6 substances with an incorrect prediction, 5 substances (2,4-Dichlorophenol, 4-Chlorophenol, Malathion, Naphthalene, Prochloraz) were less toxic to fish than to daphnids or algae, hence the AFT data would not drive the environmental risk assessment for those substances. In the case of 1 of the substances (Juglone), the AFT test was less sensitive than the FET data.

Register here: https://attendee.gotowebinar.com/register/6039364894428974605

September 24th, 2020
10:00-11:00  AM US ET// 16:00-17:00 CET

Presenter: Johanna Nyffeler, ORISE Fellow with US EPA

https://www.ascctox.org/webinar/70

The Center for Computational Toxicology & Exposure (CCTE) at U.S. EPA developed a blueprint for utilization of new approach methodologies (NAMs) in computational toxicology. The tiered approach relies on high-throughput profiling assays in the first step in characterizing the biological activity of chemicals. Chemicals are run in concentration-response in the assays for two purposes: (1) To derive a potency estimate for chemical bioactivity. This in vitro potency estimate can then be used to prioritize chemicals and/or compare the bioactivity to exposure estimates. (2) To gain information about putative mechanisms-of-actions. Depending on the mechanisms-of-action, different second and third-tier assays may then be performed to confirm hypothesized bioactivity. CCTE currently has two such profiling assays: High-throughput transcriptomics and imaging-based high-throughput phenotypic profiling (HTPP). In the webinar, I will outline our efforts to achieve these two purposes for HTPP assay. For HTPP, we are using an assay called ‘Cell Painting’, that labels multiple cellular organelles (nucleus, nucleoli, endoplasmic reticulum, golgi, actin skeleton, plasma membrane, mitochondria) to measure changes in cell morphology in response to chemical treatment. We have operationalized the assay for 384 well plates to run concentration-response screenings and extract 1300 features on a single-cell level. To date we have screened > 1200 chemicals at 8 concentrations in human U-2 OS sarcoma cells. We have derived potency estimates (i.e. phenotype altering concentrations, PACs) for all active chemicals. For 303 chemicals, in vitro-to-in vivo extrapolation was performed to compare the potency estimates to available in vivo data. For 78% of the chemicals, the HTPP potency was within two orders of magnitude from the in vivo point-of-departure. Moreover, for 72% of chemicals, HTPP was comparable or more conservative than the in vivo point-of-departure. For each active chemical, a phenotypic profile was derived from the 1300 measured features. Profiles were then compared using Pearson correlation. Among the tested chemicals were 179 chemicals with target annotations in the RefChemDB database. We could show that for two pathways (retinoic acid pathway, glucocorticoid receptor), chemicals activating the same target also display high profile similarity. Moreover, by comparing all tested chemicals to the annotated chemicals, we identified five test chemicals with high profile similarity to retinoids. Of those, four were not previously identified as modulators of the retinoic acid pathway. To summarize, our results to date indicate that the HTPP assay can be used to derive potency estimates as well as some mechanistic information that can both be used for prioritization of chemicals. This abstract does not reflect U.S. EPA policy.

Thursday, August 20th, 2020
10:00-11:00  AM US ET// 16:00-17:00 CET

Presenter: Nancy C. Baker, Leidos, Inc. and US EPA

Assessing and understanding chemical effects requires assembling information from a wide variety of sources, including millions of articles in the biomedical literature. Literature informatics approaches can help researchers make use of this information in more effective ways. We present the PubMed Abstract Sifter, a freely available literature tool from the US Environmental Protection Agency. With the Abstract Sifter, researchers can easily retrieve and triage citations from PubMed and visualize the literature landscape for a set of chemicals. The tool is supplied with template queries that facilitate the exploration of mechanistic information by using the language of Adverse Outcome Pathways (AOPs) and Key Characteristics of Carcinogens. 

This abstract does not necessarily represent US EPA policy.  

Registration is now open: https://attendee.gotowebinar.com/register/4664129833149496844

Wednesday, July 8, 2020
11:00-12:30 EDT / 17:00-18:30 CET

First speaker: Malcolm Wilkinson, Kirkstall
Title: Building more physiological models of human disease 
Quasi Vivo® Systems are a way to introduce air and media flow over in vitro cell cultures and can be used to create multi-cell or multi-organoid models for the study of disease. Current projects specifically focused on  COVID-19 include: 1) development of a model that mimics infection of by SARS-CoV-2 with natural flow of lymph and blood in the body and assess responses to the entry of SARS-CoV-2 in cells from the respiratory tract and responses on circulating blood cells from the immune system, 2) building a multi-tissue model which replicates human obesity and how different tissues communicate with each other in diseases like SARS-COVID19, and 3) improving our understanding of how cigarette smoke increases susceptibility to SARS-CoV-2 infection in the lungs and help in developing a therapy for prevention and treatment.

Second speaker: Samuel Constant, Epithelix
Title: 3D Human epithelial models to study SARS-CoV-2 pathogenesis
The respiratory system is the main entry portal of SARS-CoV-2 which infects initially and principally the airway epithelia. Epithelix has developed standardized air-liquid interface 3D human airway epithelial cultures from nasal or bronchial (MucilAir™) and small-airway (SmallAir™) origins. These epithelial models closely mimic the morphology and function of the native tissues and have been used for the development of antivirals against influenza, rhinoviruses, respiratory syncytial virus, amongst others. This talk will highlight how these reconstituted human airway epithelial models can be used to characterize viral infection kinetics, tissue-level tropism and transcriptional immune signatures induced by SARS-CoV-2. Relevance of these models for the preclinical evaluation of antiviral candidates will also be addressed in the context of repositioning of marketed drugs or evaluation of novel therapies and combinations delivered systematically or through aerosol therapy.

Registration link: https://attendee.gotowebinar.com/register/2314631813250734349

Tuesday, June 9, 2020
10:00 AM US ET / 16:00 CET

Presenter: Timothy E H Allen, Research Scientist, Willis Group, MRC Tox

Tuesday, May 26, 2020
11:00 AM US ET / 17:00 CET

Presenter: Kamel Mansouri, Integrated Laboratory Systems, Inc.

Humans are exposed to an ever-increasing number of chemicals, but only a fraction of these have been evaluated for potential risks to human health and the environment. Thus, both regulators and manufacturers need rapid and efficient approaches to evaluate the potential toxicity of thousands of chemicals already in commerce and others in development. Advances in information technology and machine learning have fostered the development of in silico approaches that leverage the relationships between chemical structures and their biological activities. However, individual predictive computational tools are associated with certain limitations, and they are only as good as the input data upon which they are built. To address these challenges, international consortia involving over 100 scientists from governmental agencies, academia, and industry were formed to collaboratively develop in silico tools for predicting chemical toxicity. These consortia have successfully concluded three projects: the Collaborative Estrogen Receptor Activity Prediction Project (CERAPP), the Collaborative Modeling Project for Androgen Receptor Activity (CoMPARA), and the Collaborative Acute Toxicity Modeling Suite (CATMoS). These projects used data from both the published literature and the ToxCast/Tox21 programs curated to meet defined quality specifications. Limitations of individual modeling approaches were overcome by establishing consensus models that leveraged each model’s strengths. The resulting consensus models have been used to screen hundreds of thousands of chemicals from the U.S. Environmental Protection Agency’s (EPA’s) DSSTox database. These models are freely available for further use through the OPEn structure-activity/property Relationship Application (OPERA), as an open-source standalone application and by querying the EPA’s CompTox chemistry Dashboard (https://comptox.epa.gov) and NTP’s Integrated Chemical Environment (https://ice.ntp.niehs.nih.gov).

Register Now: https://register.gotowebinar.com/register/4739262759308645647

References:

[1] Mansouri, K. et al. EHP: https://doi.org/10.1289/ehp.1510267

[2] Mansouri K. et al. J Cheminform: https://doi.org/10.1186/s13321-018-0263-1.

[3] Kleinstruer N.C. et al. Comput Toxicol: https://doi.org/10.1016/j.comtox.2018.08.002

[4] Mansouri, K. et al. EHP: https://doi.org/10.1289/EHP5580

Thursday, Apr 30, 2020
11:00 AM US ET / 17:00 CET

Recent advances in chemical safety assessment now allow the complete evaluation of personal care products without the use of animals. Exposure based approaches and hypothesis-driven data generation form the basis of non-animal cosmetic safety assessments.  The safety assessment approach begins with problem formulation and evaluation of existing information before considering the development of targeted testing approaches. Assessment tools, often described as New Approach Methodologies (NAMs), can include computational models and human-relevant in vitro assays which are applied in combination to provide information on ingredient hazard and risk assessment. Finally, these disparate types of information are integrated and weighted in a transparent assessment that captures uncertainty. Each presentation in this webinar will cover different aspects of this decision process.

Register Now: https://attendee.gotowebinar.com/register/5639851841859329291

Agenda

11:00 Introduction: Hypothesis led safety assessment of cosmetics,
Catherine Willett, Humane Society International

11:10  Exposure based safety assessment of cosmetics,
Corie Ellison, Procter and Gamble

11:25  The application of in silico models to support decision making in toxicology: QSAR, informatics, pathway modelling, uncertainty,
Chris Barber, Lhasa, LTD

11:40 In vitro approaches to cosmetic safety assessment
Paul Walker, Cyprotex Discovery Ltd

11:55 Integration of new approach methodologies for cosmetic safety decision making
Matt Dent, Unilever

11:10 Q & A
For more information about the project, see AFSAcollaboration.org/Cosmetics

Wednesday, March 11th, 2020
11:00 AM US ET / 17:00 CET

Presenter: Ignacio J. Tripodi, PhD Candidate in Computer Science / Interdisciplinary Quantitative Biology, University of Colorado, Boulder
Registration Link: https://attendee.gotowebinar.com/register/9200492703200567307

Government regulators and others concerned about toxic chemicals in the environment hold that a mechanistic, causal explanation of a mechanism of toxicity is strongly preferred over a statistical or machine learning-based prediction by itself. We thus present a mechanistic inference framework, which can generate hypotheses of the most likely mechanisms of toxicity for a specific chemical and cell type, using gene expression time series on human tissue and a semantically-interconnected knowledge graph. We seek enrichment in our manually-curated list of high-level mechanisms of toxicity (e.g. “Triggering of caspase-mediated apoptosis via release of cytochrome C”, or “Mitochondria-mediated toxicity by inhibition of electron transport chain”), represented as causally-linked Gene Ontology concepts.

Our knowledge representation is an extension of the PheKnowLator knowledge graph. It consists of an integration of concepts from multiple ontologies (GO, PRO, HPO, ChEBI, PATO, DOID, CL), as well as relevant concepts from Reactome, Uniprot, the cellular toxicogenomics database (CTD), and the AOP Wiki. The expression assays were obtained from the Open TG-Gates, CarcinoGenomics, and other projects, consisting of human liver, kidney, and lung, bronchial, buccal, and nasal epithelial cells exposed to a sizeable number of chemicals that elicit different mechanisms of toxicity. Both our knowledge graph and experimental transcriptomics data are human-centric. Besides predicting the most likely mechanisms of toxicity from the transcriptomics assays, we generate putative explanations based on the most significant genes at each time point with known links to their corresponding mechanism steps. This provides a transparent, possible explanation for the mechanisms of toxicity, that would help a researcher’s decision-making process and aid further experimental design. Furthermore, we were able to experimentally validate our mechanistic predictions for some chemicals without an established mechanism of toxicity.

Co-expression Network Analysis to Identify Heterogeneity Between the Breast Cancer Cell Line MCF-7 and Human Breast Cancer Tissues

Tuesday, February 25th, 2020
10:00 AM US ET / 17:00 CET

Presented by Emilie Da Silva, PhD student, Technical University of Denmark/National Research Center for the Working Environment, Denmark
Registration Link: https://attendee.gotowebinar.com/register/4278909332419455243

ABSTRACT: Testing for acute inhalation toxicity is required for chemicals manufactured or imported at tonnages ≥ 10 tons per year (Commission Regulation (EC) No 440/2008) and for biocides (Regulation (EU) No 528/2012) and plant protection products (Regulation (EU) No 283/2013) before they are allowed on the market. Currently, three OECD Test Guidelines are available (TG 403, TG 436, and TG 433), all based on the exposure of rodents to the substance of interest for up to 4 hours. The use of animals for toxicological evaluation does not come without challenges. Animal experiments are costly and time consuming. It is estimated that the turnaround time for carrying out an acute inhalation test is 3 to 4 months. Besides, using rodents in order to predict the toxicity of a compound in humans is arguable given the differences in the features of the respiratory tract. The development and the validation of alternative methods in chemico, in vitro and/or in silico are needed. To do so, the mechanistic understanding of the toxicity of inhaled chemicals is key.

An innovative in vitro method for acute inhalation toxicity will be presented. This cell-free method is based on the monitoring of lung surfactant function. Indeed, the lung surfactant layer in the alveoli is the first barrier that inhaled compounds will encounter when they reach the respiratory region in the lungs. The correlation between the inhibition of the lung surfactant function and the decrease in tidal volume in mice was shown with a variety of compounds including excipients for drug formulation, and impregnation spray products. During this webinar, the method will be introduced and explained, and case studies using different chemical groups will be presented.

Presented by Dr. Laura Escrivá, Assistant Professor, University of Valencia, Spain.
Tuesday, January 21st, 2020 at 4:00 pm CET

Available online at: https://www.ascctox.org/webinars/eVmBMbpCMaG5/61

ABSTRACT: Scientific EU criteria and guidance from ECHA and EFSA to identify substances with Endocrine Disruptor (ED) properties have recently been implemented for plant protection products and biocidal products. The ECHA/EFSA guidance, mainly addressing EATS (estrogen, androgen, thyroid, steridogenesis) modalities, provides a protocol for ED identification, which includes the evaluation of both the adverse effects and the endocrine activity, by applying weight of evidence analysis.

The assessment of the endocrine activity requires in vitro mechanistic data providing information on the mechanism through which a substance could be considered endocrine active (e.g. by binding to and activating a receptor or interfering with hormone production). Moreover, when potentially endocrine-related adverse effects and endocrine activity are identified, the biological plausibility of the link between endocrine activity and the endocrine mediated adversity should be established by a mode of action analysis. The webinar will focus on the different steps for ED identification according to the ECHA/EFSA guidance illustrating how essential it is the in vitro data for ED assessment under EU regulatory context.

Presented by Dr. Lenny Kamelia, Division of Toxicology, Wageningen University and Research, The Netherlands.
Tuesday, January 21st, 2020 at 4:00 pm CET

Available online at: https://www.ascctox.org/webinars/eVmBMbpCMaG5/61

ABSTRACT: REACH legislation requires prenatal developmental toxicity (PDT) testing for substances registered at a volume of >100 tonnes/year, which also applies to petroleum substances (PS). Given that i) PDT testing is one of the most complex, and animal- and resource-intensive regulatory requirements for substances produced at >100 tonnes/year, and ii) PS are highly complex materials (UVCBs), the development of alternative non-animal based testing strategies for PS poses huge challenges. Some PS contain high concentrations of polycyclic aromatic hydrocarbons (PAHs) and we hypothesize that PDT as observed for some PS is caused by certain types of PAH present in these products. To this purpose, DMSO-extracts of 9 PS (varying in PAH content; from 5 different product categories), 1 highly refined base oil (HRBO) (containing no PAHs), and 2 gas-to-liquid (GTL) products (devoid of PAHs) were tested in a battery of in vitro assays, including the embryonic stem cell test (EST), the zebrafish embryotoxicity test (ZET), and the aryl hydrocarbon (AhR) CALUX assay. All DMSO-extracts of the PS, but not of the HRBO and GTL, induced concentration-dependent PDT as quantified in the EST and ZET, with their potency being proportional to their 3- to 7-ring PAH content. Moreover, all PS extracts also showed AhR-mediated activity in the AhR CALUX assay, suggesting a role of the AhR in mediating the observed PDT by these substances. Combining the results of the EST, ZET, AhR CALUX assay, and the PAH content, ranked and clustered the test compounds in line with their in vivo PDT potencies. In conclusion, our battery of in vitro assays, consisting of the EST, ZET and AhR CALUX assay, is able to evaluate and differentiate the PDT potency of highly complex PS, within and among categories. The results are also in concordance with our hypothesis on the role of specific groups of PAHs present in some PS for the observed PDT induced by these substances.

Presented by Dr. Suman Chakravarti, Chief Scientific Officer, MultiCASE
December 4, 2019 at 4:00 pm CET

ABSTRACT: Use of deep learning is increasing in solving different problems in computational toxicology and QSAR methodology in general. However, it is important to recognize the strengths of deep learning techniques in order to get the best out of them.
This webinar will focus on different aspects of computational toxicity assessments that can be improved using deep learning techniques beyond just increasing the accuracy of the predictions. For example, building scalable QSARs using big data, eliminating the use of precomputed descriptors from QSARs, expanding domain of applicability of QSARs and identification of toxicity alerts using deep learning techniques. In addition, available free tools and different types of deep learning architectures that are relevant to toxicity assessments will be discussed with the aid of case studies.

December 4th Webinar, Deep Learning in Toxicity Prediction: Reasons for Use and Possible Applications, presented by Suman Chakravarti, Chief Scientific Officer, MultiCASE, has been posted online and is now available for viewing at your convenience.

Presented by Leo van Grunsven, Vrije Universiteit Brussel.
November 4, 2019 at 4:00 pm CET

ABSTRACT: Chronic liver disease is the major cause of progressive liver fibrosis which, in turn, leads to cirrhosis of the liver. One major obstacle in the development of efficient therapies is the lack of robust and representative in vitro models of human liver fibrosis to aid in understanding the basic mechanisms of the disease and in the development phase of pharmaceuticals. The aim of this presentation is to give some background on the mechanisms involved in liver fibrosis development and why our work is based on the central hypothesis that liver fibrosis in vitro cannot be studied using only hepatic stellate cells (HSCs)–the main producer of scar tissue during fibrosis. I will present and discuss some results and problems we faced while developing an in vitro liver fibrosis model. We established a model in which HepaRG cells (Biopredic) were co-cultured with either in-house isolated HSCs or in-house differentiated induced pluripotent stem cell derived HSCs. In both cases, exposure to hepatotoxins resulted in hepatocytic damage and consequent HSC activation. We now work on a model using only primary mouse HSCs and hepatocytes. I will also discuss some of our work aiming at developing more complex primary mouse co-cultures for liver disease modeling and briefly address the work of other groups that reported on such complex in vitro culture systems.

The November 4th Webinar, The pros and cons of 3D in vitro culture models of liver fibrosis, presented Prof. Leo A. van Grunsven, Vrije Universiteit Brussel, has been posted online and is now available for viewing at your convenience.

Presented by Roos Masereeuw, Div. Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Utrecht, The Netherlands.
October 22, 2019 at 4:00 pm CET

ABSTRACT: Adverse effects caused by exposure to foreign compounds, including drugs, often involve the kidney. In fact, 14 to 26% of the acute kidney injury events reported can be related to drug-induced kidney injury. Although acute damage to the kidney may be reversible, depending on severity, 30% of the patients develop structural renal dysfunction leading to chronic kidney disease eventually progressing into end-stage kidney disease. Early prediction of adverse effects such as drug interactions and renal toxicity is therefore imperative for clinical practice and for the development of new and safe drugs. Current in vitro assays do not accurately allow such prediction, predominantly due to inadequate preservation of the organs’ microenvironment. The kidney epithelium is highly polarized, and the maintenance of this polarity is critical for optimal functioning and responsiveness to environmental signals influencing cell proliferation, migration and differentiation. This presentation will provide an overview of advances in 3D cultures of human renal cells and organoids in microfluidics, and in particular kidney tubules, thereby improving physiological performance of the tissue. These microphysiological systems have great potential for drug screenings and provide novel alternative strategies for the prediction of renal drug disposition and safety assessment in a human-specific context. However, knowledge gaps in quantitative translation of renal drug disposition from microphysiological systems still exist and will be discussed.

Presented by Prof. Ellen Fritsche, Leibniz Research Institute for Environmental Medicine-Germany

August 28, 2019 at 4:00 PM CET

Presented by Dr. Lowry Curley, AxoSim Inc.
September 17, 2019 at 5:00 PM CET: