Non-animal Approaches Research Articles

Skin Sensitization Potency Prediction Based on Read-Across (RAx) Incorporating RhE-Based Testing Strategy (RTSv1)-Defined Approach: RTSv1-Based RAx.

In recent years, nonanimal approaches for skin sensitization have been developed in response to political, regulatory, and ethical demands. The reconstructed human epidermis (RhE)-based testing strategy (RTS)v1-defined approach (DA) is used to categorize skin sensitization potency. However, the RTSv1 DA alone cannot be used to predict potency based on EC3 values [the estimated concentration that produces a stimulation index of 3 in the local lymph node assay (LLNA)], and underpredictions have been reported. Read-across (RAx) can complement DA data and improve prediction confidence. Although case studies combining new approach methodology/DA data with RAx have been reported, they focus on a single target chemical and lack a comprehensive and robust strategy with well-examined reliability. This study developed a strategy incorporating the RTSv1 DA into RAx (RTSv1-based RAx) to predict skin sensitization potency, applying it to 43 chemicals. The predictive performance of RTSv1-based RAx was evaluated by comparing its predicted potency category and EC3 outcomes with those of RTSv1 DA and the LLNA. RTSv1-based RAx accurately predicted the Globally Harmonized System of Classification (GHS) subcategorization for 38 chemicals and determined the predicted EC3 values for 17 sensitizers within a fourfold range of LLNA-derived EC3 values. This study demonstrates that RTSv1-based RAx offers robust predictivity for both GHS subcategorization and predicted EC3 values, making it useful for quantitative risk assessment.

Linking EDC-laden food consumption and modern lifestyle habits with preeclampsia: A non-animal approach to identifying early diagnostic biomarkers through biochemical alterations

Preeclampsia (PE), a pregnancy complication characterized by new-onset hypertension with or without proteinuria and/or end-organ damage, and it may be influenced by exposure to endocrine-disrupting chemicals present in processed foods and modern lifestyles. This study explores the potential link using a non-animal approach to identify early diagnostic biomarkers for preeclampsia. Seventy pregnant women aged 21–41 years participated, and completed questionnaires assessing socio-demographic factors, Suboptimal Health Status Questionnaire scores for fatigue, digestive, cardiovascular, immune, and mental health issues, and exposure to endocrine-disrupting chemicals from processed food consumption and daily product use. Peripheral blood samples were analyzed for hormone profiles, complete blood count, and liver function tests (LFT). Statistical analysis revealed that mothers above 27 years old, with a Body Mass Index exceeding 32.59 Kg/m2, and a Mean Arterial Pressure of 108.5 mmHg exhibited a potential obesogenic effect on preeclampsia development. Socio-demographic factors like, lower economic class, housewife status, primiparous pregnancy, non-graduate education, and rural residence were significantly associated with results. Analysis of biochemical parameters revealed that serum creatinine, blood urea, total protein, platelet count, blood urea nitrogen, bilirubin profile, LFT profile, and thyroid profile showed potential detrimental effects on kidney, liver, muscle, and thyroid function in preeclampsia patients. Notably, PC, serum urea, bilirubin, total protein, serum glutamic-oxaloacetic transaminase (SGOT), alkaline phosphatase (ALP), and thyroid stimulating hormone (TSH) levels were significantly associated with preeclampsia in individuals reporting higher exposure to endocrine disrupting chemicals (EDCs). Minor biochemical alterations were also observed with dairy product consumption. SHS-25 analysis indicated a significant increase in fatigue, and digestive, cardiovascular, immune, and mental health-related issues in patients. Probably, biochemical alterations due to EDC exposure from processed foods and modern lifestyle habits contribute to organ dysfunction in preeclampsia. Identifying these potential biomarkers may pave the way for the development of non-invasive, early diagnostic tools for improved preeclampsia management. This research emphasizes the importance of non-animal testing methods for assessing EDC-related health risks in pregnancy and contributes to the advancement of early PE diagnosis strategies.

Building knowledge of NAMs through risk science

The 12th World Congress on Alternatives and Animal Use in the Life Sciences provided a platform for mobilizing and exchanging knowledge on the advancements in science and technology. It also provided an opportunity for experts to discuss how to accelerate the adoption of new strategies and tools. One of these recommendations advocated the need to bridge the gap between the next generation of scientists who have yet to learn about ‘New Approach Methodologies’ (NAMs) and the current generation of thought leaders who have pioneered the development and validation of these non-animal approaches to toxicological risk assessment. Consequently, a mini-course, held at Canada's University of Ottawa, was developed for students, aged 13–16 years, interested in learning about risk science and how NAMs can be used to inform human health risk assessment. This course also served as a platform for creating a virtual training roadmap, provided in this paper, thereby bringing this knowledge to a broader audience of learners who are establishing their careers in the field of risk science.

Effects of novel flame retardants tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) and triphenyl phosphate (TPhP) on function and homeostasis in human and rat pancreatic beta-cell lines

Despite the fact that environmental pollution has been implicated in the global rise of diabetes, the research on the impact of emerging pollutants such as novel flame retardants remains limited. In line with the shift towards the use of non-animal approaches in toxicological testing, this study aimed to investigate the effects of two novel flame retardants tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) and triphenyl phosphate (TPhP) in rat (INS1E) and human (NES2Y) pancreatic beta-cell lines. One-week exposure to 1 μM and 10 μM TDCIPP and TPhP altered intracellular insulin and proinsulin levels, but not the levels of secreted insulin (despite the presence of a statistically insignificant trend). The exposures also altered the protein expression of several factors involved in beta-cell metabolic pathways and signaling, including ATP citrate lyase, isocitrate dehydrogenase 1, perilipins, glucose transporters, ER stress-related factors, and antioxidant enzymes. This study has brought new and valuable insights into the toxicity of TDCIPP and TPhP on beta-cell function and revealed alterations that might impact insulin secretion after more extended exposure. It also adds to the scarce studies using in vitro pancreatic beta-cells models in toxicological testing, thereby promoting the development of non-animal testing strategy for identifying pro-diabetic effects of chemical pollutants.

Feasibility of Using a Type I IFN-Based Non-Animal Approach to Predict Vaccine Efficacy and Safety Profiles.

Animal-based tests are used for the control of vaccine quality. However, because highly purified and safe vaccines are now available, alternative approaches that can replace or reduce animal use for the assessment of vaccine outcomes must be established. In vitro tests for vaccine quality control exist and have already been implemented. However, these tests are specifically designed for some next-generation vaccines, and this makes them not readily available for testing other vaccines. Therefore, universal non-animal tests are still needed. Specific signatures of the innate immune response could represent a promising approach to predict the outcome of vaccines by non-animal methods. Type I interferons (IFNs) have multiple immunomodulatory activities, which are exerted through effectors called interferon stimulated genes (ISGs), and are one of the most important immune signatures that might provide potential candidate molecular biomarkers for this purpose. This paper will mainly examine if this idea might be feasible by analyzing all relevant published studies that have provided type I IFN-related biomarkers for evaluating the safety and efficacy profiles of vaccines using an advanced transcriptomic approach as an alternative to the animal methods. Results revealed that such an approach could potentially provide biomarkers predictive of vaccine outcomes after addressing some limitations.

Impact of a varied set of stimuli on a suite of immunological parameters within peripheral blood mononuclear cells: toward a non-animal approach for assessing immune modulation by materials intended for human use.

Introduction: In toxicology, steps are being taken towards more mechanism-focused and human relevant approaches to risk assessment, requiring new approaches and methods. Additionally, there is increasing emphasis by regulators on risk assessment of immunotoxicity. Methods: Here we present data from a peripheral blood mononuclear cell (PBMC) system whereby a varied set of stimuli, including those against the TCR and Toll-like receptors, enable readouts of cytokine and prostaglandin E2 (PGE2) production with monocyte, T cell and B cell viability, proliferation, and associated activation markers. In addition to results on the impact of the stimuli used, initial profiling data for a case study chemical, curcumin, is presented, illustrating how the system can be used to generate information on the impact of exogenous materials on three major constituent immune cell subsets for use in risk assessment and to direct follow-on studies. Results: The different stimuli drove distinct responses, not only in relation to the "quantity" of the response but also the "quality". Curcumin had a limited impact on the B cell parameters measured, with the stimuli used, and it was noted that in contrast to T cells where there was either no impact or a reduction in viability and proliferation with increasing concentration, for B cells there was a small but significant increase in both measurements at curcumin concentrations below 20µM. Similarly, whilst expression of activation markers by T cells was reduced by the highest concentration of curcumin, they were increased in B cells. Curcumin only impacted the viability of stimulated monocytes at the highest concentration and had differential impact on different activation markers. Levels of all cytokines and PGE2 were reduced at higher concentrations. Discussion: Although the platform has certain limitations, it nevertheless enables assessment of healthy baseline monocyte, T-, and B-cell responses, and scrutiny of the impact of different stimuli to detect potential immune suppression or enhancement from exogenous materials. In the case of curcumin, a pattern of responses indicative of immune suppressive / anti-inflammatory effects was detected. It is an accessible, highly modifiable system that can be used to screen materials and guide further studies, providing a holistic, integrated picture of effects.

A quantitative weight-of-evidence method for confidence assessment of adverse outcome pathway networks: A case study on chemical-induced liver steatosis

The field of chemical toxicity testing is undergoing a transition to overcome the limitations of in vivo experiments. This evolution involves implementing innovative non-animal approaches to improve predictability and provide a more precise understanding of toxicity mechanisms. Adverse outcome pathway (AOP) networks are pivotal in organizing existing mechanistic knowledge related to toxicological processes. However, these AOP networks are dynamic and require regular updates to incorporate the latest data. Regulatory challenges also persist due to concerns about the reliability of the information they offer. This study introduces a generic Weight-of-Evidence (WoE) scoring method, aligned with the tailored Bradford-Hill criteria, to quantitatively assess the confidence levels in key event relationships (KERs) within AOP networks. We use the previously published AOP network on chemical-induced liver steatosis, a prevalent form of human liver injury, as a case study. Initially, the existing AOP network is optimized with the latest scientific information extracted from PubMed using the free SysRev platform for artificial intelligence (AI)-based abstract inclusion and standardized data collection. The resulting optimized AOP network, constructed using Cytoscape, visually represents confidence levels through node size (key event, KE) and edge thickness (KERs). Additionally, a Shiny application is developed to facilitate user interaction with the dataset, promoting future updates. Our analysis of 173 research papers yielded 100 unique KEs and 221 KERs among which 72 KEs and 170 KERs, respectively, have not been previously documented in the prior AOP network or AOP-wiki. Notably, modifications in de novo lipogenesis, fatty acid uptake and mitochondrial beta-oxidation, leading to lipid accumulation and liver steatosis, garnered the highest KER confidence scores. In conclusion, our study delivers a generic methodology for developing and assessing AOP networks. The quantitative WoE scoring method facilitates in determining the level of support for KERs within the optimized AOP network, offering valuable insights into its utility in both scientific research and regulatory contexts. KERs supported by robust evidence represent promising candidates for inclusion in an in vitro test battery for reliably predicting chemical-induced liver steatosis within regulatory frameworks.

Confronting the bias towards animal experimentation (animal methods bias)

Laws and policies are in place around the world to promote the replacement and reduction of nonhuman animals in science. These principles are rooted not just in ethical considerations for animals, but also in scientific considerations regarding the limitations of using nonhuman animals to model human biology, health, and disease. New nonanimal research approaches that use human biology, cells, and data to mimic complex human physiological states and therapeutic responses have become increasingly effective and accessible, replacing the use of animals in several applications, and becoming a crucial tool for biomedical research and drug development. Despite many advantages, acceptance of these new nonanimal methods has been slow, and barriers to their broader uptake remain. One such barrier is animal methods bias, the preference for animal-based methods where they are not necessary or where animal-free methods are suitable. This bias can impact research assessments and can discourage researchers from using novel nonanimal approaches. This article provides an introductory overview of animal methods bias for the general public, reviewing evidence, exploring consequences, and discussing ongoing mitigation efforts aimed at reducing barriers in the shift away from animal use in biomedical research and testing.

Recent advances and current challenges of new approach methodologies in developmental and adult neurotoxicity testing.

Adult neurotoxicity (ANT) and developmental neurotoxicity (DNT) assessments aim to understand the adverse effects and underlying mechanisms of toxicants on the human nervous system. In recent years, there has been an increasing focus on the so-called new approach methodologies (NAMs). The Organization for Economic Co-operation and Development (OECD), together with European and American regulatory agencies, promote the use of validated alternative test systems, but to date, guidelines for regulatory DNT and ANT assessment rely primarily on classical animal testing. Alternative methods include both non-animal approaches and test systems on non-vertebrates (e.g., nematodes) or non-mammals (e.g., fish). Therefore, this review summarizes the recent advances of NAMs focusing on ANT and DNT and highlights the potential and current critical issues for the full implementation of these methods in the future. The status of the DNT in vitro battery (DNT IVB) is also reviewed as a first step of NAMs for the assessment of neurotoxicity in the regulatory context. Critical issues such as (i) the need for test batteries and method integration (from in silico and in vitro to in vivo alternatives, e.g., zebrafish, C. elegans) requiring interdisciplinarity to manage complexity, (ii) interlaboratory transferability, and (iii) the urgent need for method validation are discussed.

Next generation risk assessment: an ab initio case study to assess the systemic safety of the cosmetic ingredient, benzyl salicylate, after dermal exposure.

We performed an ab initio next-generation risk assessment (NGRA) for a fragrance ingredient, benzyl salicylate (BSal), to demonstrate how cosmetic ingredients can be evaluated for systemic toxicity endpoints based on non-animal approaches. New approach methodologies (NAMs) used to predict the internal exposure included skin absorption assays, hepatocyte metabolism, and physiologically based pharmacokinetic (PBPK) modeling, and potential toxicodynamic effects were assessed using pharmacology profiling, ToxProfiler cell stress assay, transcriptomics in HepG2 and MCF-7 cells, ReproTracker developmental and reproductive toxicology (DART) assays, and cytotoxicity assays in human kidney cells. The outcome of the NGRA was compared to that of the traditional risk assessment approach based on animal data. The identification of the toxicologically critical entity was a critical step that directed the workflow and the selection of chemicals for PBPK modeling and testing in bioassays. The traditional risk assessment and NGRA identified salicylic acid (SA) as the "toxdriver." A deterministic PBPK model for a single-day application of 1.54g face cream containing 0.5% BSal estimated the Cmax for BSal (1nM) to be much lower than that of its major in vitro metabolite, SA (93.2nM). Therefore, SA was tested using toxicodynamics bioassays. The lowest points of departure (PoDs) were obtained from the toxicogenomics assays. The interpretation of these results by two companies and methods were similar (SA only results in significant gene deregulation in HepG2 cells), but PoD differed (213μM and 10.6µM). A probabilistic PBPK model for repeated applications of the face cream estimated the highest Cmax of SA to be 630nM. The resulting margins of internal exposure (MoIE) using the PoDs were 338 and 16, which were more conservative than those derived from external exposure and in vivo PoDs (margin of safety values were 9,705). In conclusion, both traditional and ab initio NGRA approaches concluded that the daily application of BSal in a cosmetic leave-on face cream at 0.5% is safe for humans. The processing and interpretation of toxicogenomics data can lead to different PoDs, which can subsequently affect the calculation of the MoIE. This case study supports the use of NAMs in a tiered NGRA ab initio approach.

Interpretation of in vitro concentration-response data for risk assessment and regulatory decision-making: Report from the 2022 IWGT quantitative analysis expert working group meeting.

Quantitative risk assessments of chemicals are routinely performed using in vivo data from rodents; however, there is growing recognition that non-animal approaches can be human-relevant alternatives. There is an urgent need to build confidence in non-animal alternatives given the international support to reduce the use of animals in toxicity testing where possible. In order for scientists and risk assessors to prepare for this paradigm shift in toxicity assessment, standardization and consensus on in vitro testing strategies and data interpretation will need to be established. To address this issue, an Expert Working Group (EWG) of the 8th International Workshop on Genotoxicity Testing (IWGT) evaluated the utility of quantitative in vitro genotoxicity concentration-response data for risk assessment. The EWG first evaluated available in vitro methodologies and then examined the variability and maximal response of in vitro tests to estimate biologically relevant values for the critical effect sizes considered adverse or unacceptable. Next, the EWG reviewed the approaches and computational models employed to provide human-relevant dose context to in vitro data. Lastly, the EWG evaluated risk assessment applications for which in vitro data are ready for use and applications where further work is required. The EWG concluded that in vitro genotoxicity concentration-response data can be interpreted in a risk assessment context. However, prior to routine use in regulatory settings, further research will be required to address the remaining uncertainties and limitations.

Development and application of consensus in silico models for advancing high-throughput toxicological predictions

Computational toxicology models have been successfully implemented to prioritize and screen chemicals. There are numerous in silico (quantitative) structure–activity relationship ([Q]SAR) models for the prediction of a range of human-relevant toxicological endpoints, but for a given endpoint and chemical, not all predictions are identical due to differences in their training sets, algorithms, and methodology. This poses an issue for high-throughput screening of a large chemical inventory as it necessitates several models to cover diverse chemistries but will then generate data conflicts. To address this challenge, we developed a consensus modeling strategy to combine predictions obtained from different existing in silico (Q)SAR models into a single predictive value while also expanding chemical space coverage. This study developed consensus models for nine toxicological endpoints relating to estrogen receptor (ER) and androgen receptor (AR) interactions (i.e., binding, agonism, and antagonism) and genotoxicity (i.e., bacterial mutation, in vitro chromosomal aberration, and in vivo micronucleus). Consensus models were created by combining different (Q)SAR models using various weighting schemes. As a multi-objective optimization problem, there is no single best consensus model, and therefore, Pareto fronts were determined for each endpoint to identify the consensus models that optimize the multiple-criterion decisions simultaneously. Accordingly, this work presents sets of solutions for each endpoint that contain the optimal combination, regardless of the trade-off, with the results demonstrating that the consensus models improved both the predictive power and chemical space coverage. These solutions were further analyzed to find trends between the best consensus models and their components. Here, we demonstrate the development of a flexible and adaptable approach for in silico consensus modeling and its application across nine toxicological endpoints related to ER activity, AR activity, and genotoxicity. These consensus models are developed to be integrated into a larger multi-tier NAM-based framework to prioritize chemicals for further investigation and support the transition to a non-animal approach to risk assessment in Canada.

3Rs implementation in veterinary vaccine batch-release testing: Current state-of-the-art and future opportunities. A webinar and workshop report

Regulatory authorities require veterinary batch-release testing to confirm vaccine potency and safety, but these tests have traditionally relied on large numbers of laboratory animals. Advances in vaccine research and development offer increasing opportunities to replace in vivo testing, and some stakeholders have made significant progress in incorporating 3Rs elements in quality control strategies. A three-part event series entitled “3Rs Implementation in Veterinary Vaccine Batch-Release Testing: Current state-of-the-art and future opportunities” was jointly organized by the Animal-Free Safety Assessment Collaboration, HealthforAnimals, and the International Alliance of Biological Standardization. Two webinars and a workshop aimed to outline the state-of-the-art non-animal approaches for veterinary batch-release testing. The events included information on the state of the deletion of obsolete safety testing and the current initiatives implemented by European, North American, and Asian-Pacific stakeholders on 3Rs implementation and regulatory acceptance. The events contributed to a better understanding of the barriers to 3Rs implementation. Participants highlighted the need for open communication, continued collaboration between stakeholders, and international harmonization of regulatory requirements to help accelerate acceptance. Despite the challenges, the countries represented at this three-part event have shared their commitments to advancing the acceptance of alternative methods.

A non-clinical and clinical IUCLID database for 530 pharmaceuticals (part I): Methodological aspects of its development

A new IUCLID database is provided containing results from non-clinical animal studies and human information for 530 approved drugs. The database was developed by extracting data from pharmacological reviews of repeat-dose, carcinogenicity, developmental, and reproductive toxicity studies. In the database, observed and no-observed effects are linked to the respective effect levels, including information on severity/incidence and transiency/reversibility. It also includes some information on effects in humans, that were extracted from relevant sections of standard product labels of the approved drugs. The database is complemented with a specific ontology for reporting effects that was developed as an improved version of the Ontology Lookup Service's mammalian and human phenotype ontologies and includes different hierarchical levels. The developed ontology contains novel and unique standardized terms, including ontological terms for reproductive and endocrine effects. The database aims to facilitate correlation and concordance analyses based on the link between observed and no-observed effects and their respective effect levels. In addition, it offers a robust dataset on drug information for the pharmaceutical industry and research. The reported ontology supports the analyses of toxicological information, especially for reproductive and endocrine endpoints and can be used to encode legacy data or develop additional ontologies. The new database and ontology can be used to support the development of alternative non-animal approaches, to elucidate mechanisms of toxicity, and to analyse human relevance. The new IUCLID database is provided free of charge at https://iuclid6.echa.europa.eu/us-fda-toxicity-data.

Specificity of the local lymph node assay (LLNA) for skin sensitisation

The local lymph node assay (LLNA) has provided a large dataset against which performance of non-animal approaches for prediction of skin sensitisation potential and potency can be assessed. However, a recent comparison of LLNA results with human data has argued that LLNA specificity is low, with many human non-sensitisers, particularly hydrophobic chemicals, being false positives. It has been suggested that such putative false positives result from hydrophobic chemicals causing cytotoxicity, which induces irritancy, in turn driving non-specific lymphocyte proliferation. This paper finds that the apparent reduced specificity of the LLNA largely reflects differences in definitions of the boundaries between weak skin sensitisers and non-sensitisers. A small number of LLNA false positives may be due to lymphocyte proliferation without skin sensitisation, but most alleged ‘false’ positives are in fact very weak sensitisers predictable from structure-activity considerations. The evidence does not support the hypothesis for hydrophobicity-induced false positives. Moreover, the mechanistic basis is untenable. Sound LLNA data, appropriately interpreted, remain a good measure of sensitisation potency, applicable across a wide hydrophilicity-hydrophobicity range. The standard data interpretation protocol enables detection of very low levels of sensitisation, irrespective of regulatory significance, but there is scope to interpret the data to give focus on regulatory significance.

Toxicity prediction: An application of alternative testing and computational toxicology in contaminated groundwater sites in Taiwan

Groundwater contamination remains a global threat due to its toxic effects to humans and the environment. The remediation of contaminated groundwater sites can be costly, thus, identifying the priority areas of concern is important to reduce money spent on resources. In this study, we aimed to identify and rank the priority groundwater sites in a contaminated petrochemical district by combining alternative, non-animal approaches – chemical analysis, cell-based high throughput screening (HTS), and Toxicological Priority Index (ToxPi) computational toxicology tool. Groundwater samples collected from ten different sites in a contaminated district showed pollutant levels below the detection limit, however, hepatotoxic bioactivity was demonstrated in human hepatoma HepaRG cells. Integrating the pollutants information (i.e., pollutant characteristics and concentration data) with the bioactivity data of the groundwater samples, an evidence-based ranking of the groundwater sites for future remediation was established using ToxPi analysis. The currently presented combinatorial approach of screening groundwater sites for remediation purposes can further be refined by including relevant parameters, which can boost the utility of this approach for groundwater screening and future remediation.

Assessment of potential ecological risk of bisphenol A in surface waters via evaluation of alternative non-animal approaches

Recently, national regulatory agencies have announced plans to move away from animal testing through the adoption of new approach methods (NAMs) including in silico and in vitro approaches. In this study, we suggest a new approach of environmental risk assessment method for water quality criteria of bisphenol A (BPA) in the surface water of 11 rivers in Asia using an alternative toxicity method based on the in vitro-based cytotoxicity and genetic toxicity. The hazardous concentrations for 5% (HC5) were obtained from the species sensitivity distribution (SSD) curves based on the traditional acute toxicities (TAT), traditional chronic toxicities (TCT), and alterative acute toxicities (AAT) data. The HC5 values for the TAT, TCT, and AAT data were 2019.3 µg/L, 0.92 µg/L, and 0.68 µg/L, respectively. The TAT-based predicted no-effect concentration (PNECTAT), TCT-based PNEC (PNECTCT), and AAT-based PNEC (PNECAAT) values were derived as 1009.65 µg/L, 0.46 µg/L, and 0.34 µg/L, respectively. The TAT-based risk quotient (RQTAT) value of BPA was negligible (<0.1). The AAT-based RQ value (RQAAT) of BPA in Liuxi river (RQAAT = 21.94) of China showed the highest potential ecological risk of the 11 sampled rivers of Asia. In Korea, the highest potential risk assessment (RQAAT = 2.93) was recorded in Gyeonggi-do as the largest administrative region. The results may suggest that the application of AAT in the ecological risk assessment for surface water can replace the previously applied TCT approach.

P21-27 Use of non-animal approaches for pesticide safety assessment

P21-27 Use of non-animal approaches for pesticide safety assessment

Quantitative phosphoproteomics reveal cellular responses from caffeine, coumarin and quercetin in treated HepG2 cells

Protein phosphorylation is the most common type of post-translational modification where serine, threonine or tyrosine are reversibly bound to the phosphate group of ATP in a reaction catalyzed by protein kinases. Phosphorylation plays an important role in regulation of cell homeostasis, including but not limited to signal perception and transduction, gene expression and function of proteins. Protein phosphorylation happens on a fast time scale and represents an energy-efficient way for the cell to adapt to exposure to chemical stressors. To understand the cascade of cellular signaling induced by exposure to chemicals, we have exposed HepG2 cells to three chemicals with different modes of action, namely, caffeine, coumarin, and quercetin in a concentration and time response manner. Significantly upregulated and downregulated phosphosites were screened to analyze the activation/deactivation of signaling pathways by protein kinases. In total, 69, 44 and 12 signaling pathways were found enriched in caffeine, coumarin and quercetin treated cells, respectively, of which 9 pathways were co-enriched with 11 jointly responded kinases. Among identified co-responded kinases, CDK1, MAPK1 and MAPK3 play important roles in cell cycle and insulin signaling pathways. Quantitative phosphoproteomics can sensitively distinguish the effects of different chemicals on cells, allowing the assessment of chemical safety through changes in substrates and metabolic pathways at the cellular level, which is important for the development of non-animal approaches for chemical safety assessment.

Next Generation Risk Assessment of the Anti-Androgen Flutamide Including the Contribution of Its Active Metabolite Hydroxyflutamide.

In next generation risk assessment (NGRA), non-animal approaches are used to quantify the chemical concentrations required to trigger bioactivity responses, in order to assure safe levels of human exposure. A limitation of many in vitro bioactivity assays, which are used in an NGRA context as new approach methodologies (NAMs), is that toxicokinetics, including biotransformation, are not adequately captured. The present study aimed to include, as a proof of principle, the bioactivity of the metabolite hydroxyflutamide (HF) in an NGRA approach to evaluate the safety of the anti-androgen flutamide (FLU), using the AR-CALUX assay to derive the NAM point of departure (PoD). The NGRA approach applied also included PBK modelling-facilitated quantitative in vitro to in vivo extrapolation (QIVIVE). The PBK model describing FLU and HF kinetics in humans was developed using GastroPlus™ and validated against human pharmacokinetic data. PBK model-facilitated QIVIVE was performed to translate the in vitro AR-CALUX derived concentration-response data to a corresponding in vivo dose-response curve for the anti-androgenicity of FLU, excluding and including the activity of HF (-HF and +HF, respectively). The in vivo benchmark dose 5% lower confidence limits (BMDL05) derived from the predicted in vivo dose-response curves for FLU, revealed a 440-fold lower BMDL05 when taking the bioactivity of HF into account. Subsequent comparison of the predicted BMDL05 values to the human therapeutic doses and historical animal derived PoDs, revealed that PBK modelling-facilitated QIVIVE that includes the bioactivity of the active metabolite is protective and provides a more appropriate PoD to assure human safety via NGRA, whereas excluding this would potentially result in an underestimation of the risk of FLU exposure in humans.