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.
•The nominal concentration of a chemical is not similarly proportional to the dose at the target site across in vitro assays.•Chemicals can distribute to different components of the in vitro assay.•The distribution of a chemical in vitro depends on the physicochemical properties of the test chemical and in vitro assay setup.•Distribution processes can be modelled using rate constants or partition coefficients.•A number of in silico models have been developed to predict the in vitro distribution of test chemicals.•In silico models predicting the distribution of test chemicals in vitro have different chemical and biological applicability domains.
In vitro assays are normally conducted in plastic multiwell plates open to exchange with the ambient air. The concentration of test substances freely available to cells is often not known, can change ...over time, and is difficult to measure in the small volumes in microplates. However, even a well-characterized toxicological response is of limited value if it cannot be linked to a well-defined exposure level. The aim of this study was to develop and apply an approach for determining time-resolved freely dissolved concentrations of semivolatile and hydrophobic organic chemicals (SVHOCs) in in vitro assays: (1) free fractions were measured by a new medium dilution method and (2) time-resolved loss curves were obtained by measurements of total concentrations in 96-well plates during incubations at 37 °C. Headspace solid-phase microextraction was used as an analytical technique for 24 model chemicals spanning 6 chemical groups and 4–5 orders of magnitude in K ow and K aw. Free fractions were >30% for chemicals with log K ow < 3.5 and then decreased with increasing log K ow. Medium concentrations declined significantly (>50%) within 24 h of incubation for all 20 chemicals having log K ow > 4 or log K aw > −3.5 in serum-free medium. Losses of chemicals were lower for medium containing 10% fetal bovine serum, most significantly for chemicals with log K ow > 4. High crossover to neighboring wells also was observed below log K ow of 4 and log K aw of −3.5. Sealing the well plates had limited effect on the losses but clearly reduced crossover. The high losses and crossover of most tested chemicals question the suitability of multiwell plates for in vitro testing of SVHOCs and call for (1) test systems that minimize losses, (2) methods to control in vitro exposure, (3) analytical confirmation of exposure, and (4) exposure control and confirmation being included in good in vitro reporting standards.
Current acceptable chemical exposure levels (e.g., tolerable daily intake) are mainly based on animal experiments, which are costly, time-consuming, considered non-ethical by many, and may poorly ...predict adverse outcomes in humans.
To evaluate a method using human in vitro data and biological modeling to calculate an acceptable exposure level through a case study on 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) developmental neurotoxicity (DNT).
We reviewed the literature on in vitro assays studying BDE-47-induced DNT. Using the most sensitive endpoint, we derived a point of departure using a mass-balance in vitro disposition model and benchmark dose modeling for a 5% response (BMC05) in cells. We subsequently used a pharmacokinetic model of gestation and lactation to estimate administered equivalent doses leading to four different metrics of child brain concentration (i.e., average prenatal, average postnatal, average overall, and maximum concentration) equal to the point of departure. The administered equivalent doses were translated into tolerable daily intakes using uncertainty factors. Finally, we calculated biomonitoring equivalents for maternal serum and compared them to published epidemiological studies of DNT.
We calculated a BMC05 of 164 μg/kg of cells for BDE-47 induced alteration of differentiation in neural progenitor cells. We estimated administered equivalent doses of 0.925–3.767 μg/kg/day in mothers, and tolerable daily intakes of 0.009–0.038 μg/kg/day (composite uncertainty factor: 100). The lowest derived biomonitoring equivalent was 19.75 ng/g lipids, which was consistent with reported median (0.9–23 ng/g lipids) and geometric mean (7.02–26.9 ng/g lipids) maternal serum concentrations from epidemiological studies.
This case study supports using in vitro data and biological modeling as a viable alternative to animal testing to derive acceptable exposure levels.
The aim of the EU FP7 Predict-IV project was to improve the predictivity of in vitro assays for unwanted effects of drugs after repeated dosing. The project assessed the added benefit of integrating ...long-lived in vitro organotypic cell systems with ‘omics’ technologies and in silico modelling, including systems biology and pharmacokinetic assessments. RPTEC/TERT1 kidney cells, primary rat and human hepatocytes, HepaRG liver cells and 2D and 3D primary brain cultures were dosed daily or every other day for 14days to a selection of drugs varying in their mechanism of pharmacological action. Since concentration–effect relationships not only depend on the activity of the drug or the sensitivity of the target, but also on the distribution of compounds in the in vitro system, the concentration of a selection of drugs in cells, microtitre plate plastic and medium was measured over time. Results, reviewed in this paper, indicate that lipophilic drugs bind significantly to plastic labware. A few drugs, including less lipophilic drugs, bind to cell-attachment matrices. Chemicals that reach high concentrations in cells, including cyclosporin A and amiodarone, significantly accumulate over time after repeated dosing, partly explaining their increased toxicity after repeated dosing, compared to a single dose.
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•This paper reviews work done within WP3 of the EU FP7 Predict-IV project.•RPTEC/TERT1, HepaRG, primary hepatocytes and brain cultures were considered.•Medium dosed with drugs was refreshed daily or every other day for 14days.•Drug concentrations over time were measured and modelled in various system phases.•Drugs differentially accumulate in cells in repeatedly-dosed in vitro assays.
•Taxonomic keys for identification of viper species.•Venom composition, mode of action of viper toxins.•Protocol for the clinical management of viper envenomation.•Future perspectives are discussed.
...Snakebites in Europe are mostly due to bites from Viperidae species of the genus Vipera. This represents a neglected public health hazard with poorly defined incidence, morbidity and mortality. In Europe, fourteen species of “true vipers” (subfamily Viperinae) are present, eleven of which belong to the genus Vipera. Amongst these, the main medically relevant species due to their greater diffusion across Europe and the highest number of registered snakebites are six, namely: Vipera ammodytes, V. aspis, V. berus, V. latastei, V. seoanei and V. ursinii. Generally speaking, viper venom composition is characterised by many different toxin families, like phospholipases A2, snake venom serine proteases, snake venom metalloproteases, cysteine‐rich secretory proteins, C‐type lectins, disintegrins, haemorrhagic factors and coagulation inhibitors. A suspected snakebite is often associated with severe pain, erythema, oedema and, subsequently, the onset of an ecchymotic area around one or two visible fang marks. In the field, the affected limb should be immobilised and mildly compressed with a bandage, which can then be removed once the patient is being treated in hospital. The clinician should advise the patient to remain calm to reduce blood circulation and, therefore, decrease the spread of the toxins. In the case of pain, an analgesic therapy can be administered, the affected area can be treated with hydrogen peroxide or clean water. However, anti-inflammatory drugs and disinfection with alcohol or alcoholic substances should be avoided. For each patient, clinical chemistry and ECG are always a pre-requisite as well as the evaluation of the tetanus immunisation status and for which immunisation may be provided if needed.
The treatment of any clinical complication, due to the envenomation, does not differ from treatments of emergency nature. Antivenom is recommended when signs of systemic envenomation exist or in case of advanced local or systemic progressive symptoms. Recommendations for future work concludes.
The aim of this review is to support clinicians for the clinical management of viper envenomation, through taxonomic keys for main species identification, description of venom composition and mode of action of known toxins and provide a standardised clinical protocol and antivenom administration.
Abstract 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.
Environmental contaminants pose serious health threats to marine megafauna species, yet methods defining exposure threshold limits are lacking. Here, a three-pillar chemical risk assessment framework ...is presented based on (1) species- and chemical-specific lifetime bioaccumulation modelling, (2) non-destructive in vitro and in vivo toxicity threshold assessment, and (3) chemical risk quantification. We used the effects of cadmium (Cd) in green sea turtles (Chelonia mydas) as a proof of concept to evaluate the quantitative mechanistic modelling approach. A physiologically-based kinetic (PBK) model simulated Cd tissue concentrations (liver, kidney, muscle, fat, brain, scute, and ‘rest of the body’) in C.mydas. The validated PBK model then translated species-specific in vitro results to in vivo effects. The results showed that the resilience of C.mydas towards Cd kidney toxicity is age-dependent and differs with changing physiology and feeding ecology. Using the model in reverse mode, a steady-state exposure threshold of 0.1 µg/g dry weight Cd in forage was derived and compared to real-world exposure scenarios. Three out of the four globally distinct C.mydas populations assessed are exposed to Cd levels above this threshold limit. This approach can be adapted to other marine species and chemicals to prioritize measures for managing potentially harmful chemical exposures.
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•Lifetime physiologically based kinetic (PBK) model of cadmium (Cd) in green sea turtle (C. mydas).•Quantitative in vitro-in vivo extrapolation (QIVIVE) modelling for risk assessment using species-specific C.mydas cell models.•Predicted dietary toxicity threshold for C. mydas is 0.1 μg/g dry weight Cd for risk assessment.•The majority of C. mydas foraging populations are at risk.
Amphibian populations are undergoing a global decline worldwide. Such decline has been attributed to their unique physiology, ecology, and exposure to multiple stressors including chemicals, ...temperature, and biological hazards such as fungi of the Batrachochytrium genus, viruses such as Ranavirus, and habitat reduction. There are limited toxicity data for chemicals available for amphibians and few quantitative structure-activity relationship (QSAR) models have been developed and are publicly available. Such QSARs provide important tools to assess the toxicity of chemicals particularly in a data poor context. QSARs provide important tools to assess the toxicity of chemicals particularly when no toxicological data are available. This manuscript provides a description and validation of a regression-based QSAR model to predict, in a quantitative manner, acute lethal toxicity of aromatic chemicals in tadpoles of the Japanese brown frog (Rana japonica). QSAR models for acute median lethal molar concentrations (LC50-12 h) of waterborne chemicals using the Monte Carlo method were developed. The statistical characteristics of the QSARs were described as average values obtained from five random distributions into training and validation sets. Predictions from the model gave satisfactory results for the overall training set (R2 = 0.72 and RMSE = 0.33) and were even more robust for the validation set (R2 = 0.96 and RMSE = 0.11). Further development of QSAR models in amphibians, particularly for other life stages and species, are discussed.
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•Few QSAR models are available for the prediction of chemical toxicity in amphibians.•QSAR models have been developed here to predict acute lethal toxicity of aromatic chemicals in Rana japonica.•Satisfactory prediction results for the training set and robust results for validation set•QSAR model development for other life stages and amphibian species is proposed.
Current points of departure used to derive health-based guidance values for deoxynivalenol (DON) are based on studies in laboratory animals. Here, an animal-free testing approach was adopted in which ...a reverse dosimetry physiologically based kinetic (PBK) modeling is used to predict in vivo dose response curves for DON’s effects on intestinal pro-inflammatory cytokine secretion and intestinal bile acid reabsorption in humans from concentration–effect relationships for DON in vitro. The calculated doses for inducing a 5% added effect above the background level (ED5) of DON for increasing IL-1β secretion in intestinal tissue and for increasing the amounts in the colon lumen of glycochenodeoxycholic acid (GCDCA) were 246 and 36 μg/kg bw/day, respectively. These in vitro–in silico-derived ED5 values were compared to human dietary DON exposure levels, indicating that the risk of DON’s effects on these end points occurring in various human populations cannot be excluded. This in vitro–in silico approach provides a novel testing strategy for hazard and risk assessment without using laboratory animals.
Current climate trends are likely to expand the geographic distribution of the toxigenic microalgae and concomitant phycotoxins, making intoxications by such toxins a global phenomenon. Among various ...phycotoxins, saxitoxin (STX) acts as a neurotoxin that might cause severe neurological symptoms in mammals following consumptions of contaminated seafood. To derive a point of departure (POD) for human health risk assessment upon acute neurotoxicity induced by oral STX exposure, a physiologically based kinetic (PBK) modeling-facilitated quantitative in vitro to in vivo extrapolation (QIVIVE) approach was employed. The PBK models for rats, mice, and humans were built using parameters from the literature, in vitro experiments, and in silico predictions. Available in vitro toxicity data for STX were converted to in vivo dose–response curves via the PBK models established for these three species, and POD values were derived from the predicted curves and compared to reported in vivo toxicity data. Interspecies differences in acute STX toxicity between rodents and humans were found, and they appeared to be mainly due to differences in toxicokinetics. The described approach resulted in adequate predictions for acute oral STX exposure, indicating that new approach methodologies, when appropriately integrated, can be used in a 3R-based chemical risk assessment paradigm.
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