RESUMEN
Next Generation Risk Assessment (NGRA) is an exposure-led approach to safety assessment that uses New Approach Methodologies (NAMs). Application of NGRA has been largely restricted to assessments of consumer use of cosmetics and is not currently implemented in occupational safety assessments, e.g. under EU REACH. By contrast, a large proportion of regulatory worker safety assessments are underpinned by toxicological studies using experimental animals. Consequently, occupational safety assessment represents an area that would benefit from increasing application of NGRA to safety decision making. Here, a workflow for conducting NGRA under an occupational safety context was developed, which is illustrated with a case study chemical; sodium 2-hydroxyethane sulphonate (sodium isethionate or SI). Exposures were estimated using a standard occupational exposure model following a comprehensive life cycle assessment of SI and considering factory-specific data. Outputs of this model were then used to estimate internal exposures using a Physiologically Based Kinetic (PBK) model, which was constructed with SI specific Absorption, Distribution, Metabolism and Excretion (ADME) data. PBK modelling indicated a worst-case plasma maximum concentration (Cmax) of 0.8⯵M across the SI life cycle. SI bioactivity was assessed in a battery of NAMs relevant to systemic, reproductive, and developmental toxicity; a cell stress panel, high throughput transcriptomics in three cell lines (HepG2, HepaRG and MCF-7 cells), pharmacological profiling and specific assays relating to developmental toxicity (Reprotracker and devTOX quickPredict). Points of Departure (PoDs) for SI ranged from 104 to 5044⯵M. Cmax values obtained from PBK modelling of occupational exposures to SI were compared with PoDs from the bioactivity assays to derive Bioactivity Exposure Ratios (BERs) which demonstrated the safety for workers exposed to SI under current levels of factory specific risk management. In summary, the tiered and iterative workflow developed here represents an opportunity for integrating non animal approaches for a large subset of substances for which systemic worker safety assessment is required. Such an approach could be followed to ensure that animal testing is only conducted as a "last resort" e.g. under EU REACH.
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Exposición Profesional , Medición de Riesgo/métodos , Humanos , Exposición Profesional/normas , Exposición Profesional/efectos adversos , Seguridad Química/métodos , Animales , Salud Laboral , Modelos Biológicos , Pruebas de Toxicidad/métodos , Ácidos Sulfónicos/toxicidadRESUMEN
With the move away from safety testing assessment based on data generated in experimental animals the concept of Next Generation Risk Assessment (NGRA) has arisen which instead uses data from in silico and in vitro models. A key uncertainty in risk assessment is the actual dose of test chemical at the target site, and therefore surrogate dose metrics, such as nominal concentration in test media are used to describe in vitro effect (or no-effect) doses. The reliability and accuracy of the risk assessment therefore depends largely on our ability to understand and characterise the relationship between the dose metrics used and the actual biologically effective dose at the target site. The objective of this publication is to use 40 case study chemicals to illustrate how in vitro dose considerations can be applied to characterise the "true dose" and build confidence in the understanding of the biologically effective dose in in vitro test systems for the determination e.g. points of departure (PoDs) for NGRA. We propose a workflow that can be applied to assess whether the nominal test concentration can be considered a conservative dose metric for use in NGRA. The workflow examines the implications of volatility, stability, hydrophobicity, binding to plastic and serum, solubility, and the potential use of in silico models for some of these parameters. For the majority of the case study chemicals we found that the use of nominal concentrations in risk assessment would result in conservative decision making. However, for serval chemicals a potential for underestimation of the risk in humans in vivo based on in vitro nominal effect concentrations was identified, and approaches for refinement by characterisation of the actual effect concentration are proposed.
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Relación Dosis-Respuesta a Droga , Pruebas de Toxicidad , Medición de Riesgo/métodos , Pruebas de Toxicidad/métodos , Humanos , Animales , Simulación por Computador , Flujo de TrabajoRESUMEN
As part of the safety assessment of salicylate esters in cosmetics, we developed a metabolism factor based on in vitro to in vivo extrapolation (IVIVE) to provide a better estimation of the aggregate internal exposure to the common metabolite, salicylic acid. Optimal incubation conditions using human liver S9 were identified before measuring salicylic acid formation from 31 substances. Four control substances, not defined as salicylic esters but which could be mistaken as such due to their nomenclature, did not form salicylic acid. For the remaining substances, higher in vitro intrinsic clearance (CLint, in vitro) values generally correlated with lower LogP values. A "High-Throughput Pharmacokinetic" (HTPK) model was used to extrapolate CLint, in vitro values to human in vivo clearance and half-lives. The latter were used to calculate the percentage of substance metabolised to salicylic acid in 24 h in vivo following human exposure to the ester, i.e. the "metabolism factor". The IVIVE model correctly reproduced the observed elimination rate of 3 substances using in silico or in vitro input parameters. For other substances, in silico only-based predictions generally resulted in lower metabolism factors than when in vitro values for plasma binding and liver S9 CLint, in vitro were used. Therefore, in vitro data input provides the more conservative metabolism factors compared to those derived using on in silico input. In conclusion, these results indicate that not all substances contribute equally (or at all) to the systemic exposure to salicylic acid. Therefore, we propose a realistic metabolism correction factor by which the potential contribution of salicylate esters to the aggregate consumer exposure to salicylic acid from cosmetic use can be estimated.
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Ésteres , Ácido Salicílico , Humanos , Ácido Salicílico/farmacocinética , Ácido Salicílico/metabolismo , Cosméticos , Modelos Biológicos , Administración Cutánea , Hígado/metabolismo , Hígado/efectos de los fármacos , Semivida , Piel/metabolismo , Piel/efectos de los fármacos , Simulación por Computador , Absorción CutáneaRESUMEN
Estimating human exposure in the safety assessment of chemicals is crucial. Physiologically based kinetic (PBK) models which combine information on exposure, physiology, and chemical properties, describing the absorption, distribution, metabolism, and excretion (ADME) processes of a chemical, can be used to calculate internal exposure metrics such as maximum concentration and area under the concentration-time curve in plasma or tissues of a test chemical in next-generation risk assessment. This article demonstrates the development of PBK models for 3 UV filters, specifically octyl methoxycinnamate, octocrylene, and 4-methylbenzylidene camphor. The models were parameterized entirely based on data obtained from in vitro and/or in silico methods in a bottom-up modeling approach and then validated based on human dermal pharmacokinetic (PK) data. The 3 UV filters are "difficult to test" in in vitro test systems due to high lipophilicity, high binding affinity for proteins, and nonspecific binding, for example, toward plastic. This research work presents critical considerations in ADME data generation, interpretation, and parameterization to assure valid PBK model development to increase confidence in using PBK modeling to help make safety decisions in the absence of human PK data. The developed PBK models of the 3 chemicals successfully simulated the plasma concentration profiles of clinical PK data following dermal application, indicating the reliability of the ADME data generated and the parameters determined. The study also provides insights and lessons learned for characterizing ADME and developing PBK models for highly lipophilic and protein-bound chemicals in the future.
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Modelos Biológicos , Humanos , Reproducibilidad de los Resultados , Cinética , Medición de Riesgo , Técnicas In VitroRESUMEN
Nominal concentrations (CNom) in cell culture media are routinely used to define concentration-effect relationships in the in vitro toxicology. The actual concentration in the medium (CMedium) can be affected by adsorption processes, evaporation, or degradation of chemicals. Therefore, we measured the total and free concentration of 12 chemicals, covering a wide range of lipophilicity (logâ¯KOW -0.07-6.84), in the culture medium (CMedium) and cells (CCell) after incubation with Balb/c 3T3 cells for up to 48 h. Measured values were compared to predictions using an as yet unpublished in silico mass balance model that combined relevant equations from similar models published by others. The total CMedium for all chemicals except tamoxifen (TAM) were similar to the CNom. This was attributed to the cellular uptake of TAM and accumulation into lysosomes. The free (i.e., unbound) CMedium for the low/no protein binding chemicals were similar to the CNom, whereas values of all moderately to highly protein-bound chemicals were less than 30% of the CNom. Of the 12 chemicals, the two most hydrophilic chemicals, acetaminophen (APAP) and caffeine (CAF), were the only ones for which the CCell was the same as the CNom. The CCell for all other chemicals tended to increase over time and were all 2- to 274-fold higher than CNom. Measurements of CCytosol, using a digitonin method to release cytosol, compared well with CCell (using a freeze-thaw method) for four chemicals (CAF, APAP, FLU, and KET), indicating that both methods could be used. The mass balance model predicted the total CMedium within 30% of the measured values for 11 chemicals. The free CMedium of all 12 chemicals were predicted within 3-fold of the measured values. There was a poorer prediction of CCell values, with a median overprediction of 3- to 4-fold. In conclusion, while the number of chemicals in the study is limited, it demonstrates the large differences between CNom and total and free CMedium and CCell, which were also relatively well predicted by the mass balance model.
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Acetaminofén , Técnicas de Cultivo de Célula , Ratones , Animales , Interacciones Hidrofóbicas e Hidrofílicas , Unión ProteicaRESUMEN
An important question in toxicological risk assessment is whether non-animal new approach methodologies (NAMs) can be used to make safety decisions that are protective of human health, without being overly conservative. In this work, we propose a core NAM toolbox and workflow for conducting systemic safety assessments for adult consumers. We also present an approach for evaluating how protective and useful the toolbox and workflow are by benchmarking against historical safety decisions. The toolbox includes physiologically based kinetic (PBK) models to estimate systemic Cmax levels in humans, and 3 bioactivity platforms, comprising high-throughput transcriptomics, a cell stress panel, and in vitro pharmacological profiling, from which points of departure are estimated. A Bayesian model was developed to quantify the uncertainty in the Cmax estimates depending on how the PBK models were parameterized. The feasibility of the evaluation approach was tested using 24 exposure scenarios from 10 chemicals, some of which would be considered high risk from a consumer goods perspective (eg, drugs that are systemically bioactive) and some low risk (eg, existing food or cosmetic ingredients). Using novel protectiveness and utility metrics, it was shown that up to 69% (9/13) of the low risk scenarios could be identified as such using the toolbox, whilst being protective against all (5/5) the high-risk ones. The results demonstrated how robust safety decisions could be made without using animal data. This work will enable a full evaluation to assess how protective and useful the toolbox and workflow are across a broader range of chemical-exposure scenarios.
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Cosméticos , Adulto , Teorema de Bayes , Benchmarking , Humanos , Medición de Riesgo , Flujo de TrabajoRESUMEN
Abiotic stability of chemicals is not routinely tested prior to performing in vitro bioassays, although abiotic degradation can reduce the concentration of test chemicals leading to the formation of active or inactive transformation products, which may lead to misinterpretation of bioassay results. A high-throughput workflow was developed to measure the abiotic stability of 22 test chemicals in protein-rich aqueous media under typical bioassay conditions at 37 °C for 48 h. These test chemicals were degradable in the environment according to a literature review. The chemicals were extracted from the exposure media at different time points using a novel 96-pin solid-phase microextraction. The conditions were varied to differentiate between various reaction mechanisms. For most hydrolyzable chemicals, pH-dependent degradation in phosphate-buffered saline indicated that acid-catalyzed hydrolysis was less important than reactions with hydroxide ions. Reactions with proteins were mainly responsible for the depletion of the test chemicals in the media, which was simulated by bovine serum albumin (BSA) and glutathione (GSH). 1,2-Benzisothiazol-3(2H)-one, 2-methyl-4-isothiazolinone, and l-sulforaphane reacted almost instantaneously with GSH but not with BSA, indicating that GSH is a good proxy for reactivity with electrophilic amino acids but may overestimate the actual reaction with three-dimensional proteins. Chemicals such as hydroquinones or polyunsaturated chemicals are prone to autoxidation, but this reaction is difficult to differentiate from hydrolysis and could not be simulated by the oxidant N-bromosuccinimide. Photodegradation played a minor role because cells are exposed in incubators in the dark and simulations with high light intensities did not yield realistic degradation. Stability predictions from various in silico prediction models for environmental conditions can give initial indications of the stability but were not always consistent with the experimental stability in bioassays. As the presented workflow can be performed in high throughput under realistic bioassay conditions, it can be used to provide an experimental database for developing bioassay-specific stability prediction models.
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Bioensayo , Microextracción en Fase Sólida , Bioensayo/métodos , Hidrólisis , Fotólisis , Albúmina Sérica Bovina/química , Agua/químicaRESUMEN
Combined with in vitro bioactivity data, physiologically based kinetic (PBK) models has increasing applications in next generation risk assessment for animal-free safety decision making. A tiered framework of building PBK models for such application has been developed with increasing complexity and refinements, as model parameters determined in silico, in vitro, and with human pharmacokinetic data become progressively available. PBK modelling has been widely applied for oral/intravenous administration, but less so on topically applied chemicals. Therefore, building PBK models for topical applications and characterizing their uncertainties in the tiered approach is critical to safety decision making. The purpose of this study was to assess the confidence of PBK modelling of topically applied chemicals following the tiered framework, using non-animal methods derived parameters. Prediction of maximum plasma concentration (Cmax) and area under the curve were compared to observed kinetics from published dermal clinical studies for five chemicals (diclofenac, salicylic acid, coumarin, nicotine, caffeine). A bespoke Bayesian statistical model was developed to describe the distributions of Cmax errors between the predicted and observed data. We showed a general trend that confidence in model predictions increases when more quality in vitro data, particularly those on hepatic clearance and dermal absorption, are available as model input. The overall fold error distributions are useful for characterizing model uncertainty. We concluded that by identifying and quantifying the uncertainties in the tiered approach, we can increase the confidence in using PBK modelling to help make safety decisions on topically applied chemicals in the absence of human pharmacokinetic data.
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Hígado , Modelos Biológicos , Teorema de Bayes , Humanos , Cinética , Medición de Riesgo/métodos , IncertidumbreRESUMEN
Using the chemical doxorubicin (DOX), the objective of the present study was to evaluate the impact of dose metrics selection in the new approach method of integrating physiologically-based kinetic (PBK) modelling and relevant human cell-based assays to inform a priori the point of departure for human health risk. We reviewed the literature on the clinical consequences of DOX treatment to identify dosing scenarios with no or mild cardiotoxicity observed. Key concentrations of DOX that induced cardiomyocyte toxicity in vitro were derived from studies of our own and others. A human population-based PBK model of DOX was developed and verified against pharmacokinetic data. The model was then used to predict plasma and extracellular and intracellular heart concentrations of DOX under selected clinical settings and compared with in vitro outcomes, based on several dose metrics: Cmax (maximum concentration) or AUC (area under concentration-time curve) in free or total form of DOX. We found when using in vitro assays to predict cardiotoxicity for DOX, AUC is a better indicator. Our study illustrates that when appropriate dose metrics are used, it is possible to combine PBK modelling with in vitro-derived toxicity information to define margins of safety and predict low-risk human exposure levels.
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Antibióticos Antineoplásicos/farmacocinética , Doxorrubicina/farmacocinética , Modelos Biológicos , Medición de Riesgo/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antibióticos Antineoplásicos/administración & dosificación , Antibióticos Antineoplásicos/sangre , Línea Celular , Doxorrubicina/administración & dosificación , Doxorrubicina/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Miocardio/metabolismo , Adulto JovenRESUMEN
Nominal effect concentrations from in vitro toxicity assays may lead to inaccurate estimations of in vivo toxic doses because the nominal concentration poorly reflects the concentration at the molecular target in cells in vitro, which is responsible for initiating effects and can be referred to as the biologically effective dose. Chemicals can differentially distribute between in vitro assay compartments, including serum constituents in exposure medium, microtitre plate plastic, headspace and extracellular matrices. The partitioning of test chemicals to these extracellular compartments reduces the concentration at the molecular target. Free concentrations in medium and cell-associated concentrations are considered better proxies of the biologically effective dose. This paper reviews the mechanisms by which test chemicals distribute between in vitro assay compartments, and also lists the physicochemical properties driving the extent of this distribution. The mechanisms and physicochemical properties driving the distribution of test chemical in vitro help explain the makeup of mass balance models that estimate free concentrations and cell-associated concentrations in in vitro toxicity assays. A thorough understanding of the distribution processes and assumptions underlying these mass balance models helps define chemical and biological applicability domains of individual models, as well as provide a perspective on how to improve model predictivity and quantitative in vitro-in vivo extrapolations.
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Bioensayo , Modelos Biológicos , Pruebas de Toxicidad , Animales , Simulación por Computador , Humanos , Preparaciones Farmacéuticas/administración & dosificación , Preparaciones Farmacéuticas/química , Preparaciones Farmacéuticas/metabolismoRESUMEN
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.
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Cosméticos , Cumarinas/toxicidad , Pruebas de Toxicidad , Animales , Biología Computacional , Simulación por Computador , Seguridad de Productos para el Consumidor , Composición Familiar , Humanos , Medición de RiesgoRESUMEN
The freely dissolved concentration in the assay medium (Cfree) and the total cellular concentration (Ccell) are essential input parameters for quantitative in vitro-to-in vivo extrapolations (QIVIVE), but available prediction tools for Cfree and Ccell have not been sufficiently validated with experimental data. In this study, medium-water distribution ratios (DFBS/w) and cell-water distribution ratios (Dcell/w) for four different cells lines were determined experimentally for 12 neutral and five ionizable chemicals. Literature data for seven organic acids were added to the dataset, leading to 24 chemicals in total. A mass balance model based on bovine serum albumin-water (DBSA/w) and liposome-water distribution ratios (Dlip/w) of the chemicals was used to calculate DFBS/w and Dcell/w. For all neutral and basic test chemicals, the mass balance model predicted DFBS/w and Dcell/w within a factor of 3 and 3.4, respectively, indicating that existing models can reliably predict Cfree and Ccell for these chemicals. For organic acids, a further refinement of the model will be required as large deviations between modeled and measured binding to assay medium and cells of up to a factor of 370 were found. Furthermore, saturation of medium proteins should be further explored for organic acids and neutral chemicals with moderate hydrophobicity.
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Compuestos Orgánicos , Albúmina Sérica Bovina , Bioensayo , Interacciones Hidrofóbicas e Hidrofílicas , AguaRESUMEN
Next Generation Risk Assessment (NGRA) is a procedure that integrates new approach methodologies (NAMs) to assure safety of a product without generating data from animal testing. One of the major challenges in the application of NGRA to consumer products is how to extrapolate from the in vitro points of departure (PoDs) to the human exposure level associated with product use. To bridge the gap, physiologically based kinetic (PBK) modelling is routinely used to predict systemic exposure (Cmax or AUC) from external exposures. A novel framework was developed for assessing the exposure of new ingredients in dermally applied products based on the construction of PBK models describing consumer habits and practices, formulation type, and ADME (absorption, distribution, metabolism and excretion) properties exclusively obtained from NAMs. This framework aims to quantify and reduce the uncertainty in predictions and is closely related to the risk assessment process (i.e., is the margin of safety sufficient to cover the uncertainties in the extrapolation between the in vitro and in vivo toxicodynamics and toxicokinetics?). Coumarin, caffeine, and sulforaphane in four product types (kitchen cleaner liquid, face cream, shampoo, and body lotion) were selected to exemplify how this framework could be used in practise. Our work shows initial levels of the framework provide a conservative estimate of Cmax in most cases which can be refined using sensitivity analysis to inform the choice of follow-up in vitro experiments. These case studies show the framework can increase confidence in use of PBK predictions for safety assessment.
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Seguridad de Productos para el Consumidor , Modelos Biológicos , Administración Cutánea , Cafeína/sangre , Cafeína/farmacocinética , Simulación por Computador , Cosméticos/farmacocinética , Cumarinas/sangre , Cumarinas/farmacocinética , Detergentes/farmacocinética , Humanos , Isotiocianatos/sangre , Isotiocianatos/farmacocinética , Medición de Riesgo , Absorción Cutánea , SulfóxidosRESUMEN
The nominal concentration is generally used to express concentration-effect relationships in in vitro toxicity assays. However, the nominal concentration does not necessarily represent the exposure concentration responsible for the observed effect. Surfactants accumulate at interphases and likely sorb to in vitro system components such as serum protein and well plate plastic. The extent of sorption and the consequences of this sorption on in vitro readouts is largely unknown for these chemicals. The aim of this study was to demonstrate the effect of sorption to in vitro components on the observed cytotoxic potency of benzalkonium chlorides (BAC) varying in alkyl chain length (6-18 carbon atoms, C6-18) in a basal cytotoxicity assay with the rainbow trout gill cell line (RTgill-W1). Cells were exposed for 48 h in 96-well plates to increasing concentration of BACs in exposure medium containing 0, 60 µM bovine serum albumin (BSA) or 10% fetal bovine serum (FBS). Before and after exposure, BAC concentrations in exposure medium were analytically determined. Based on freely dissolved concentrations at the end of the exposure, median effect concentrations (EC50) decreased with increasing alkyl chain length up to 14 carbons. For BAC with alkyl chains of 12 or more carbons, EC50's based on measured concentrations after exposure in supplement-free medium were up to 25-times lower than EC50's calculated using nominal concentrations. When BSA or FBS was added to the medium, a decrease in cytotoxic potency of up to 22 times was observed for BAC with alkyl chains of eight or more carbons. The results of this study emphasize the importance of expressing the in vitro readouts as a function of a dose metric that is least influenced by assay setup to compare assay sensitivities and chemical potencies.
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Compuestos de Benzalconio/farmacología , Animales , Compuestos de Benzalconio/química , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Estructura Molecular , Oncorhynchus mykiss , Relación Estructura-ActividadRESUMEN
In vitro high-throughput non-depletive quantitation of chemicals in biofluids is of growing interest in many areas. Some of the challenges facing researchers include the limited volume of biofluids, rapid and high-throughput sampling requirements, and the lack of reliable methods. Coupled to the above, growing interest in the monitoring of kinetics and dynamics of miniaturized biosystems has spurred the demand for development of novel and revolutionary methodologies for analysis of biofluids. The applicability of solid-phase microextraction (SPME) is investigated as a potential technology to fulfill the aforementioned requirements. As analytes with sufficient diversity in their physicochemical features, nicotine, N,N-Diethyl-meta-toluamide, and diclofenac were selected as test compounds for the study. The objective was to develop methodologies that would allow repeated non-depletive sampling from 96-well plates, using 100 µL of sample. Initially, thin film-SPME was investigated. Results revealed substantial depletion and consequent disruption in the system. Therefore, new ultra-thin coated fibers were developed. The applicability of this device to the described sampling scenario was tested by determining the protein binding of the analytes. Results showed good agreement with rapid equilibrium dialysis. The presented method allows high-throughput analysis using small volumes, enabling fast reliable free and total concentration determinations without disruption of system equilibrium.
RESUMEN
Risk assessment methodologies in toxicology have remained largely unchanged for decades. The default approach uses high dose animal studies, together with human exposure estimates, and conservative assessment (uncertainty) factors or linear extrapolations to determine whether a specific chemical exposure is 'safe' or 'unsafe'. Although some incremental changes have appeared over the years, results from all new approaches are still judged against this process of extrapolating high-dose effects in animals to low-dose exposures in humans. The US National Research Council blueprint for change, entitled Toxicity Testing in the 21st Century: A Vision and Strategy called for a transformation of toxicity testing from a system based on high-dose studies in laboratory animals to one founded primarily on in vitro methods that evaluate changes in normal cellular signalling pathways using human-relevant cells or tissues. More recently, this concept of pathways-based approaches to risk assessment has been expanded by the description of 'Adverse Outcome Pathways' (AOPs). The question, however, has been how to translate this AOP/TT21C vision into the practical tools that will be useful to those expected to make safety decisions. We have sought to provide a practical example of how the TT21C vision can be implemented to facilitate a safety assessment for a commercial chemical without the use of animal testing. To this end, the key elements of the TT21C vision have been broken down to a set of actions that can be brought together to achieve such a safety assessment. Such components of a pathways-based risk assessment have been widely discussed, however to-date, no worked examples of the entire risk assessment process exist. In order to begin to test the process, we have taken the approach of examining a prototype toxicity pathway (DNA damage responses mediated by the p53 network) and constructing a strategy for the development of a pathway based risk assessment for a specific chemical in a case study mode. This contribution represents a 'work-in-progress' and is meant to both highlight concepts that are well-developed and identify aspects of the overall process which require additional development. To guide our understanding of what a pathways-based risk assessment could look like in practice, we chose to work on a case study chemical (quercetin) with a defined human exposure and to bring a multidisciplinary team of chemists, biologists, modellers and risk assessors to work together towards a safety assessment. Our goal was to see if the in vitro dose response for quercetin could be sufficiently understood to construct a TT21C risk assessment without recourse to rodent carcinogenicity study data. The data presented include high throughput pathway biomarkers (p-H2AX, p-ATM, p-ATR, p-Chk2, p53, p-p53, MDM2 and Wip1) and markers of cell-cycle, apoptosis and micronuclei formation, plus gene transcription in HT1080 cells. Eighteen point dose response curves were generated using flow cytometry and imaging to determine the concentrations that resulted in significant perturbation. NOELs and BMDs were compared to the output from biokinetic modelling and the potential for in vitro to in vivo extrapolation explored. A first tier risk assessment was performed comparing the total quercetin concentration in the in vitro systems with the predicted total quercetin concentration in plasma and tissues. The shortcomings of this approach and recommendations for improvement are described. This paper therefore describes the current progress in an ongoing research effort aimed at providing a pathways-based, proof-of-concept in vitro-only safety assessment for a consumer use product.
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Técnicas In Vitro , Modelos Biológicos , Quercetina/toxicidad , Transducción de Señal/efectos de los fármacos , Pruebas de Toxicidad/métodos , Toxicología/métodos , Alternativas a las Pruebas en Animales , Animales , Línea Celular Tumoral , Simulación por Computador , Seguridad de Productos para el Consumidor , Daño del ADN , Relación Dosis-Respuesta a Droga , Ensayos Analíticos de Alto Rendimiento , Humanos , Técnicas In Vitro/tendencias , Nivel sin Efectos Adversos Observados , Quercetina/farmacocinética , Medición de Riesgo , Factores de Riesgo , Biología de Sistemas , Pruebas de Toxicidad/tendencias , Toxicología/tendencias , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Challenges to improve toxicological risk assessment to meet the demands of the EU chemical's legislation, REACH, and the EU 7th Amendment of the Cosmetics Directive have accelerated the development of non-animal based methods. Unfortunately, uncertainties remain surrounding the power of alternative methods such as in vitro assays to predict in vivo dose-response relationships, which impedes their use in regulatory toxicology. One issue reviewed here, is the lack of a well-defined dose metric for use in concentration-effect relationships obtained from in vitro cell assays. Traditionally, the nominal concentration has been used to define in vitro concentration-effect relationships. However, chemicals may differentially and non-specifically bind to medium constituents, well plate plastic and cells. They may also evaporate, degrade or be metabolized over the exposure period at different rates. Studies have shown that these processes may reduce the bioavailable and biologically effective dose of test chemicals in in vitro assays to levels far below their nominal concentration. This subsequently hampers the interpretation of in vitro data to predict and compare the true toxic potency of test chemicals. Therefore, this review discusses a number of dose metrics and their dependency on in vitro assay setup. Recommendations are given on when to consider alternative dose metrics instead of nominal concentrations, in order to reduce effect concentration variability between in vitro assays and between in vitro and in vivo assays in toxicology.
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Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Modelos Biológicos , Pruebas de Toxicidad/métodos , Toxicología/métodos , Alternativas a las Pruebas en Animales , Animales , Simulación por Computador , Humanos , Técnicas In Vitro/normas , Reproducibilidad de los Resultados , Medición de Riesgo , Factores de Riesgo , Biología de Sistemas , Factores de Tiempo , Pruebas de Toxicidad/normas , Toxicología/normasRESUMEN
PURPOSE: Green tea is thought to possess many beneficial effects on human health. However, the extent of green tea polyphenol biotransformation may affect its proposed therapeutic effects. Catechol-O-methyltransferase (COMT), the enzyme responsible for polyphenolic methylation, has a common polymorphism in the genetic code at position 158 reported to result in a 40% reduction in enzyme activity in in vitro studies. The current preliminary study was designed to investigate the impact of COMT genotype on green tea catechin absorption and metabolism in humans. METHODS: Twenty participants (10 of each homozygous COMT genotype) were recruited, and plasma concentration profiles were produced for epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), epicatechin (EC) and 4'-O-methyl EGCG after 1.1 g of Sunphenon decaffeinated green tea extract (836 mg green tea catechins), with a meal given after 60 min. RESULTS: For the entire group, EGCG, EGC, EC, ECG and 4'-O-methyl EGCG reached maximum concentrations of 1.09, 0.41, 0.33, 0.16 and 0.08 µM at 81.5, 98.5, 99.0, 85.5 and 96.5 min, respectively. Bimodal curves were observed for the non-gallated green tea catechins EGC and EC as opposed to single-peaked curves for the gallated green tea catechins EGCG and ECG. No significant parametric differences between COMT genotype groups were found. CONCLUSIONS: In conclusion, the COMT Val(158/108)Met does not appear to have a dramatic influence on EGCG absorption and elimination. However, further pharmacokinetic research is needed to substantiate these findings.