RESUMO
Per- and poly-fluoroalkyl substances (PFAS) have a wide range of elimination half-lives (days to years) in humans, thought to be in part due to variation in proximal tubule reabsorption. While human biomonitoring studies provide important data for some PFAS, renal clearance (CLrenal) predictions for hundreds of PFAS in commerce requires experimental studies with in vitro models and physiologically-based in vitro-to-in vivo extrapolation (IVIVE). Options for studying renal proximal tubule pharmacokinetics include cultures of renal proximal tubule epithelial cells (RPTECs) and/or microphysiological systems. This study aimed to compare CLrenal predictions for PFAS using in vitro models of varying complexity (96-well plates, static 24-well Transwells and a fluidic microphysiological model, all using human telomerase reverse transcriptase-immortalized and OAT1-overexpressing RPTECs combined with in silico physiologically-based IVIVE. Three PFAS were tested: one with a long half-life (PFOS) and two with shorter half-lives (PFHxA and PFBS). PFAS were added either individually (5 µM) or as a mixture (2 µM of each substance) for 48 h. Bayesian methods were used to fit concentrations measured in media and cells to a three-compartmental model to obtain the in vitro permeability rates, which were then used as inputs for a physiologically-based IVIVE model to estimate in vivo CLrenal. Our predictions for human CLrenal of PFAS were highly concordant with available values from in vivo human studies. The relative values of CLrenal between slow- and faster-clearance PFAS were most highly concordant between predictions from 2D culture and corresponding in vivo values. However, the predictions from the more complex model (with or without flow) exhibited greater concordance with absolute CLrenal. Overall, we conclude that a combined in vitro-in silico workflow can predict absolute CLrenal values, and effectively distinguish between PFAS with slow and faster clearance, thereby allowing prioritization of PFAS with a greater potential for bioaccumulation in humans.
Assuntos
Simulação por Computador , Fluorocarbonos , Túbulos Renais Proximais , Modelos Biológicos , Humanos , Fluorocarbonos/farmacocinética , Túbulos Renais Proximais/metabolismo , Meia-Vida , Taxa de Depuração Metabólica , Fluxo de Trabalho , Eliminação Renal , Poluentes Ambientais/farmacocinética , Poluentes Ambientais/metabolismo , Células Epiteliais/metabolismoRESUMO
Microphysiological systems are an emerging area of in vitro drug development, and their independent evaluation is important for wide adoption and use. The primary goal of this study was to test reproducibility and robustness of a renal proximal tubule microphysiological system, OrganoPlate 3-lane 40, as an in vitro model for drug transport and toxicity studies. This microfluidic model was compared with static multiwell cultures and tested using several human renal proximal tubule epithelial cell (RPTEC) types. The model was characterized in terms of the functional transport for various tubule-specific proteins, epithelial permeability of small molecules (cisplatin, tenofovir, and perfluorooctanoic acid) versus large molecules (fluorescent dextrans, 60-150 kDa), and gene expression response to a nephrotoxic xenobiotic. The advantages offered by OrganoPlate 3-lane 40 as compared with multiwell cultures are the presence of media flow, albeit intermittent, and increased throughput compared with other microfluidic models. However, OrganoPlate 3-lane 40 model appeared to offer only limited (eg, MRP-mediated transport) advantages in terms of either gene expression or functional transport when compared with the multiwell plate culture conditions. Although OrganoPlate 3-lane 40 can be used to study cellular uptake and direct toxic effects of small molecules, it may have limited utility for drug transport studies. Overall, this study offers refined experimental protocols and comprehensive comparative data on the function of RPETCs in traditional multiwell culture and microfluidic OrganoPlate 3-lane 40, information that will be invaluable for the prospective end-users of in vitro models of the human proximal tubule.
Assuntos
Túbulos Renais Proximais , Sistemas Microfisiológicos , Humanos , Reprodutibilidade dos Testes , Estudos Prospectivos , RimRESUMO
New approach methodologies (NAMs) that do not use experimental animals are, in certain settings, entirely appropriate for assuring the safety of chemical ingredients, although regulatory adoption has been slow. In this opinion article we discuss how scientific advances that utilize NAMs to certify systemic safety are available now and merit broader acceptance within the framework of next generation risk assessments (NGRA).
Assuntos
Alternativas aos Testes com Animais , Segurança Química , Animais , Medição de RiscoRESUMO
Much has been written and said about the promise and excitement of microphysiological systems, miniature devices that aim to recreate aspects of human physiology on a chip. The rapid explosion of the offerings and persistent publicity placed high expectations on both product manufacturers and regulatory agencies to adopt the data. Inevitably, discussions of where this technology fits in chemical testing paradigms are ongoing. Some end-users became early adopters, whereas others have taken a more cautious approach because of the high cost and uncertainties of their utility. Here, we detail the experience of a public-private collaboration established for testing of diverse microphysiological systems. Collectively, we present a number of considerations on practical aspects of using microphysiological systems in the context of their applications in decision-making. Specifically, future end-users need to be prepared for extensive on-site optimization and have access to a wide range of imaging and other equipment. We reason that cells, related reagents, and the technical skills of the research staff, not the devices themselves, are the most critical determinants of success. Extrapolation from concentration-response effects in microphysiological systems to human blood or oral exposures, difficulties with replicating the whole organ, and long-term functionality remain as critical challenges. Overall, we conclude that it is unlikely that a rodent- or human-equivalent model is achievable through a finite number of microphysiological systems in the near future; therefore, building consensus and promoting the gradual incorporation of these models into tiered approaches for safety assessment and decision-making is the sensible path to wide adoption.
Assuntos
Dispositivos Lab-On-A-Chip , HumanosRESUMO
New Approach Methodologies (NAMs) promise to offer a unique opportunity to enable human-relevant safety decisions to be made without the need for animal testing in the context of exposure-driven Next Generation Risk Assessment (NGRA). Protecting human health against the potential effects a chemical may have on embryo-foetal development and/or aspects of reproductive biology using NGRA is particularly challenging. These are not single endpoint or health effects and risk assessments have traditionally relied on data from Developmental and Reproductive Toxicity (DART) tests in animals. There are numerous Adverse Outcome Pathways (AOPs) that can lead to DART, which means defining and developing strict testing strategies for every AOP, to predict apical outcomes, is neither a tenable goal nor a necessity to ensure NAM-based safety assessments are fit-for-purpose. Instead, a pragmatic approach is needed that uses the available knowledge and data to ensure NAM-based exposure-led safety assessments are sufficiently protective. To this end, the mechanistic and biological coverage of existing NAMs for DART were assessed and gaps to be addressed were identified, allowing the development of an approach that relies on generating data relevant to the overall mechanisms involved in human reproduction and embryo-foetal development. Using the knowledge of cellular processes and signalling pathways underlying the key stages in reproduction and development, we have developed a broad outline of endpoints informative of DART. When the existing NAMs were compared against this outline to determine whether they provide comprehensive coverage when integrated in a framework, we found them to generally cover the reproductive and developmental processes underlying the traditionally evaluated apical endpoint studies. The application of this safety assessment framework is illustrated using an exposure-led case study.
RESUMO
Many substances for which consumer safety risk assessments need to be conducted are not associated with specific toxicity modes of action, but rather exhibit nonspecific toxicity leading to cell stress. In this work, a cellular stress panel is described, consisting of 36 biomarkers representing mitochondrial toxicity, cell stress, and cell health, measured predominantly using high content imaging. To evaluate the panel, data were generated for 13 substances at exposures consistent with typical use-case scenarios. These included some that have been shown to cause adverse effects in a proportion of exposed humans and have a toxicological mode-of-action associated with cellular stress (eg, doxorubicin, troglitazone, and diclofenac), and some that are not associated with adverse effects due to cellular stress at human-relevant exposures (eg, caffeine, niacinamide, and phenoxyethanol). For each substance, concentration response data were generated for each biomarker at 3 timepoints. A Bayesian model was then developed to quantify the evidence for a biological response, and if present, a credibility range for the estimated point of departure (PoD) was determined. PoDs were compared with the plasma Cmax associated with the typical substance exposures, and indicated a clear differentiation between "low" risk and "high" risk chemical exposure scenarios. Developing robust methods to characterize the in vitro bioactivity of xenobiotics is an important part of non-animal safety assessment. The results presented in this work show that the cellular stress panel can be used, together with other new approach methodologies, to identify chemical exposures that are protective of consumer health.
Assuntos
Qualidade de Produtos para o Consumidor , Medição de Risco/métodos , Estresse Fisiológico , Animais , Teorema de Bayes , Biomarcadores , Características da Família , Humanos , XenobióticosRESUMO
Next-Generation Risk Assessment is defined as an exposure-led, hypothesis-driven risk assessment approach that integrates new approach methodologies (NAMs) to assure safety without the use of animal testing. These principles were applied to a hypothetical safety assessment of 0.1% coumarin in face cream and body lotion. For the purpose of evaluating the use of NAMs, existing animal and human data on coumarin were excluded. Internal concentrations (plasma Cmax) were estimated using a physiologically based kinetic model for dermally applied coumarin. Systemic toxicity was assessed using a battery of in vitro NAMs to identify points of departure (PoDs) for a variety of biological effects such as receptor-mediated and immunomodulatory effects (Eurofins SafetyScreen44 and BioMap Diversity 8 Panel, respectively), and general bioactivity (ToxCast data, an in vitro cell stress panel and high-throughput transcriptomics). In addition, in silico alerts for genotoxicity were followed up with the ToxTracker tool. The PoDs from the in vitro assays were plotted against the calculated in vivo exposure to calculate a margin of safety with associated uncertainty. The predicted Cmax values for face cream and body lotion were lower than all PoDs with margin of safety higher than 100. Furthermore, coumarin was not genotoxic, did not bind to any of the 44 receptors tested and did not show any immunomodulatory effects at consumer-relevant exposures. In conclusion, this case study demonstrated the value of integrating exposure science, computational modeling and in vitro bioactivity data, to reach a safety decision without animal data.