Human exposure to synthetic endocrine disrupting chemicals (S-EDCs) is generally negligible as compared to natural compounds with higher or comparable endocrine activity: how to evaluate the risk of the S-EDCs?

Theoretically, both synthetic endocrine disrupting chemicals (S-EDCs) and natural (exogenous and endogenous) endocrine disrupting chemicals (N-EDCs) can interact with endocrine receptors and disturb hormonal balance. However, compared to endogenous hormones, S-EDCs are only weak partial agonists with receptor affinities several orders of magnitude lower. Thus, to elicit observable effects, S-EDCs require considerably higher concentrations to attain sufficient receptor occupancy or to displace natural hormones and other endogenous ligands. Significant exposures to exogenous N-EDCs may result from ingestion of foods such as soy-based diets, green tea and sweet mustard. While their potencies are lower as compared to natural endogenous hormones, they usually are considerably more potent than S-EDCs. Effects of exogenous N-EDCs on the endocrine system were observed at high dietary intakes. A causal relation between their mechanism of action and these effects is established and biologically plausible. In contrast, the assumption that the much lower human exposures to S-EDCs may induce observable endocrine effects is not plausible. Hence, it is not surprising that epidemiological studies searching for an association between S-EDC exposure and health effects have failed. Regarding testing for potential endocrine effects, a scientifically justified screen should use in vitro tests to compare potencies of S-EDCs with those of reference N-EDCs. When the potency of the S-EDC is similar or smaller than that of the N-EDC, further testing in laboratory animals and regulatory consequences are not warranted.

Human exposure to synthetic endocrine disrupting chemicals (S-EDCs) is generally negligible as compared to natural compounds with higher or comparable endocrine activity. How to evaluate the risk of the S-EDCs?

Theoretically, both synthetic endocrine-disrupting chemicals (S-EDCs) and natural (exogenous and endogenous) endocrine-disrupting chemicals (N-EDCs) can interact with endocrine receptors and disturb hormonal balance. However, compared to endogenous hormones, S-EDCs are only weak partial agonists with receptor affinities several orders of magnitude lower than S-EDCs. Thus, to elicit observable effects, S-EDCs require considerably higher concentrations to attain sufficient receptor occupancy or to displace natural hormones and other endogenous ligands. Significant exposures to exogenous N-EDCs may result from ingestion of foods such as soy-based diets, green tea, and sweet mustard. While their potencies are lower as compared to natural endogenous hormones, they usually are considerably more potent than S-EDCs. Effects of exogenous N-EDCs on the endocrine system were observed at high dietary intakes. A causal relation between their mechanism of action and these effects is established and biologically plausible. In contrast, the assumption that the much lower human exposures to S-EDCs may induce observable endocrine effects is not plausible. Hence, it is not surprising that epidemiological studies searching for an association between S-EDC exposure and health effects have failed. Regarding testing for potential endocrine effects, a scientifically justified screen should use in vitro tests to compare potencies of S-EDCs with those of reference N-EDCs. When the potency of the S-EDC is similar or smaller than that of the N-EDC, further testing in laboratory animals and regulatory consequences are not warranted.

Influence of in Vitro Assay Setup on the Apparent Cytotoxic Potency of Benzalkonium Chlorides.

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.

Prediction of in vivo developmental toxicity of all-trans-retinoic acid based on in vitro toxicity data and in silico physiologically based kinetic modeling.

The use of laboratory animals for toxicity testing in chemical safety assessment meets increasing ethical, economic and legislative constraints. The development, validation and application of reliable alternatives for in vivo toxicity testing are therefore urgently needed. In order to use toxicity data obtained from in vitro assays for risk assessment, in vitro concentration-response data need to be translated into in vivo dose-response data that are needed to obtain points of departure for risk assessment, like a benchmark dose (BMD). In the present study, we translated in vitro concentration-response data of the retinoid all-trans-retinoic acid (ATRA), obtained in the differentiation assay of the embryonic stem cell test, into in vivo dose-response data using a physiologically based kinetic model for rat and human that is mainly based on kinetic model parameter values derived using in vitro techniques. The predicted in vivo dose-response data were used for BMD modeling, and the obtained BMDL10 values [lower limit of the 95 % confidence interval on the BMD at which a benchmark response equivalent to a 10 % effect size (BMR10) is reached (BMD10)] for rat were compared with BMDL10 values derived from in vivo developmental toxicity data in rats reported in the literature. The results show that the BMDL10 values from predicted dose-response data differ about sixfold from the BMDL10 values obtained from in vivo data, pointing at the feasibility of using a combined in vitro-in silico approach for defining a point of departure for toxicological risk assessment.

Regulatory acceptance and use of the Extended One Generation Reproductive Toxicity Study within Europe.

The two-generation study (OECD TG 416) is the standard requirement within REACH to test reproductive toxicity effects of chemicals with production volumes >100 tonnes. This test is criticized in terms of scientific relevance and animal welfare. The Extended One Generation Reproductive Toxicity Study (EOGRTS), incorporated into the OECD test guidelines in 2011 (OECD TG 443) has the potential to replace TG 416, while using only one generation of rats and being more informative. However, its regulatory acceptance proved challenging. This article reconstructs the process of regulatory acceptance and use of the EOGRTS and describes drivers and barriers influencing the process. The findings derive from literature research and expert interviews. A distinction is made between three sub-stages; The stage of Formal Incorporation of the EOGRTS into OECD test guidelines was stimulated by retrospective analyses on the value of the second generation (F2), strong EOGRTS advocates, animal welfare concern and changing US and EU chemicals legislation; the stage of Actual Regulatory Acceptance within REACH was challenged by legal factors and ongoing scientific disputes, while the stage of Use by Industry is influenced by uncertainty of registrants about regulatory acceptance, high costs, the risk of false positives and the manageability of the EOGRTS.

A novel hypothesis for an alkaline phosphatase 'rescue' mechanism in the hepatic acute phase immune response.

The liver isoform of the enzyme alkaline phosphatase (AP) has been used classically as a serum biomarker for hepatic disease states such as hepatitis, steatosis, cirrhosis, drug-induced liver injury, and hepatocellular carcinoma. Recent studies have demonstrated a more general anti-inflammatory role for AP, as it is capable of dephosphorylating potentially deleterious molecules such as nucleotide phosphates, the pathogenic endotoxin lipopolysaccharide (LPS), and the contact clotting pathway activator polyphosphate (polyP), thereby reducing inflammation and coagulopathy systemically. Yet the mechanism underlying the observed increase in liver AP levels in circulation during inflammatory insults is largely unknown. This paper hypothesizes an immunological role for AP in the liver and the potential of this system for damping generalized inflammation along with a wide range of ancillary pathologies. Based on the provided framework, a mechanism is proposed in which AP undergoes transcytosis in hepatocytes from the canalicular membrane to the sinusoidal membrane during inflammation and the enzyme's expression is upregulated as a result. Through a tightly controlled, nucleotide-stimulated negative feedback process, AP is transported in this model as an immune complex with immunoglobulin G by the asialoglycoprotein receptor through the cell and secreted into the serum, likely using the receptor's State 1 pathway. The subsequent dephosphorylation of inflammatory stimuli by AP and uptake of the circulating immune complex by endothelial cells and macrophages may lead to decreased inflammation and coagulopathy while providing an early upstream signal for the induction of a number of anti-inflammatory gene products, including AP itself.

Leukemia from dermal exposure to cyclophosphamide among nurses in The Netherlands: quantitative assessment of the risk.

Several studies showed that oncology nurses are exposed to antineoplastic drugs via the skin during daily activities. Several antineoplastic drugs (including cyclophosphamide) have been classified as carcinogenic to humans. This study aims to assess the leukemia risk of occupational exposure to cyclophosphamide. Average task frequencies from the population of oncology nurses in the Netherlands and task-based dermal exposure intensities were used to calculate oncology nurses' dermal exposure levels. A dermal absorption model in combination with a physiologically based pharmacokinetic model was used to assess the delivered dose of cyclophosphamide and its active metabolites in the bone marrow. This delivered dose was subsequently related to pharmacodynamic and epidemiological information from a longitudinal study with cyclophosphamide-treated patients to estimate the excess lifetime leukemia risk at age 80 for Dutch oncology nurses after 40 years of exposure to cyclophosphamide. The excess lifetime leukemia risk at age 80 of an exposed oncology nurse after 40 years of dermal exposure to cyclophosphamide was estimated to be 1.04 per million oncology nurses. This risk could potentially increase to a maximum of 154 per million if a nurse performs all cyclophosphamide-related tasks with the maximum frequency (as observed in this population) and is exposed to maximum exposure intensities for each task without using protective gloves for 40 years. This study indicates that the risk of an oncology nurse in a Dutch hospital with an average dermal exposure to cyclophosphamide is well below the maximum tolerable risk of one extra death from cancer per 250 deaths after 40 years of occupational exposure, and that this level is not exceeded in a worst-case scenario.

Regulatory acceptance and use of 3R models for pharmaceuticals and chemicals: expert opinions on the state of affairs and the way forward.

Pharmaceuticals and chemicals are subjected to regulatory safety testing accounting for approximately 25% of laboratory animal use in Europe. This testing meets various objections and has led to the development of a range of 3R models to Replace, Reduce or Refine the animal models. However, these models must overcome many barriers before being accepted for regulatory risk management purposes. This paper describes the barriers and drivers and options to optimize this acceptance process as identified by two expert panels, one on pharmaceuticals and one on chemicals. To untangle the complex acceptance process, the multilevel perspective on technology transitions is applied. This perspective defines influences at the micro-, meso- and macro level which need alignment to induce regulatory acceptance of a 3R model. This paper displays that there are many similar mechanisms within both sectors that prevent 3R models from becoming accepted for regulatory risk assessment and management. Shared barriers include the uncertainty about the value of the new 3R models (micro level), the lack of harmonization of regulatory requirements and acceptance criteria (meso level) and the high levels of risk aversion (macro level). In optimizing the process commitment, communication, cooperation and coordination are identified as critical drivers.

Quantifying processes determining the free concentration of phenanthrene in Basal cytotoxicity assays.

Difficulties may arise when extrapolating in vitro derived toxicity data to in vivo toxicity data because of the high variability and occasional low sensitivity of in vitro results. Differences in the free concentration of a test compound between in vitro and in vivo systems and between different in vitro systems may in part explain this variability and sensitivity difference. The aim of this study was to determine what assay components influence the free concentration of phenanthrene in a Balb/c 3T3 and RTgill-W1 MTT assay. Partition coefficients of phenanthrene to serum, well plate plastic, cells, and headspace were measured and subsequently used to model the free concentration of the compound in vitro. The estimated free concentration was compared to the free concentration measured in the assays using solid phase microextraction (SPME). Results indicate that the free concentration of phenanthrene, a relatively volatile and hydrophobic compound, is significantly reduced in a typical in vitro setup as it binds to matrices such as serum protein and well plate plastic. A reduction in free concentration due to increasing serum protein levels is accompanied by an increase in the median effect concentration (EC(50)) and can be modeled, with the exception of evaporation, using the partition coefficients of the compound to assay components.

Transport of chlorpromazine in the Caco-2 cell permeability assay: a kinetic study.

The intestinal transport of compounds can be measured in vitro with Caco-2 cell monolayers. We took a closer look at the exposure and fate of a chemical in the Caco-2 cell assay, including the effect of protein binding. Transport of chlorpromazine (CPZ) was measured in the absorptive and secretory direction, with and without albumin basolaterally. Samples were taken from medium, cells, and well plastic. For the secretory transport experiments with albumin, the free CPZ concentration at the start of the experiment was measured by negligible depletion-solid phase microextraction (nd-SPME). Recovery of CPZ from the medium was low, especially in the absorptive transport direction. CPZ was found in the cells (≤20%) and bound to the well plastic (≤25%), and 94% of CPZ was bound to albumin. An initial lag phase was observed, which was likely caused by partitioning of CPZ between the donor concentration and the Caco-2 cells; after 20 min, transport of CPZ to the receiver compartment was linear. The low recovery and the test compound found both inside the Caco-2 cells and bound to the well plastic complicate the calculation of the fraction transported and render reliable estimates of permeability constants impossible. For a chemical like chlorpromazine, which is hydrophobic in its neutral form, but in general also for more lipophilic compounds, the Caco-2 cell assay might not be straightforward, and a more detailed study into the fate and exposure of the test compound might be needed to arrive at meaningful data for transport and permeability.

Evaluation of research activities and research needs to increase the impact and applicability of alternative testing strategies in risk assessment practice.

The present paper aims at identifying strategies to increase the impact and applicability of alternative testing strategies in risk assessment. To this end, a quantitative and qualitative literature evaluation was performed on (a) current research efforts in the development of in vitro methods aiming for alternatives to animal testing, (b) the possibilities and limitations of in vitro methods for regulatory purposes and (c) the potential of physiologically-based kinetic (PBK) modeling to improve the impact and applicability of in vitro methods in risk assessment practice. Overall, the evaluation showed that the focus of state-of-the-art research activities does not seem to be optimally directed at developing in vitro alternatives for those endpoints that are most animal-demanding, such as reproductive and developmental toxicity, and carcinogenicity. A key limitation in the application of in vitro alternatives to such systemic endpoints is that in vitro methods do not provide so-called points of departure, necessary for regulators to set safe exposure limits. PBK-modeling could contribute to overcoming this limitation by providing a method that allows extrapolation of in vitro concentration-response curves to in vivo dose-response curves. However, more proofs of principle are required.

Improving the quality of toxicology and environmental health systematic reviews: What journal editors can do.

Systematic reviews are fast increasing in prevalence in the toxicology and environmental health literature. However, how well these complex research projects are being conducted and reported is unclear. Since editors have an essential role in ensuring the scientific quality of manuscripts being published in their journals, a workshop was convened where editors, systematic review practitioners, and research quality control experts could discuss what editors can do to ensure the systematic reviews they publish are of sufficient scientific quality. Interventions were explored along four themes: setting standards; reviewing protocols; optimizing editorial workflows; and measuring the effectiveness of editorial interventions. In total, 58 editorial interventions were proposed. Of these, 26 were shortlisted for being potentially effective, and 5 were prioritized as short-term actions that editors could relatively easily take to improve the quality of published systematic reviews. Recent progress in improving systematic reviews is summarized, and outstanding challenges to further progress are highlighted.

Decrease of intracellular pH as possible mechanism of embryotoxicity of glycol ether alkoxyacetic acid metabolites.

Embryotoxicity of glycol ethers is caused by their alkoxyacetic acid metabolites, but the mechanism underlying the embryotoxicity of these acid metabolites is so far not known. The present study investigates a possible mechanism underlying the embryotoxicity of glycol ether alkoxyacetic acid metabolites using the methoxyacetic acid (MAA) metabolite of ethylene glycol monomethyl ether as the model compound. The results obtained demonstrate an MAA-induced decrease of the intracellular pH (pH(i)) of embryonic BALB/c-3T3 cells as well as of embryonic stem (ES)-D3 cells, at concentrations that affect ES-D3 cell differentiation. These results suggest a mechanism for MAA-mediated embryotoxicity similar to the mechanism of embryotoxicity of the drugs valproic acid and acetazolamide (ACZ), known to decrease the pH(i)in vivo, and therefore used as positive controls. The embryotoxic alkoxyacetic acid metabolites ethoxyacetic acid, butoxyacetic acid and phenoxyacetic acid also caused an intracellular acidification of BALB/c-3T3 cells at concentrations that are known to inhibit ES-D3 cell differentiation. Two other embryotoxic compounds, all-trans-retinoic acid and 5-fluorouracil, did not decrease the pH(i) of embryonic cells at concentrations that affect ES-D3 cell differentiation, pointing at a different mechanism of embryotoxicity of these compounds. MAA and ACZ induced a concentration-dependent inhibition of ES-D3 cell differentiation, which was enhanced by amiloride, an inhibitor of the Na(+)/H(+)-antiporter, corroborating an important role of the pH(i) in the embryotoxic mechanism of both compounds. Together, the results presented indicate that a decrease of the pH(i) may be the mechanism of embryotoxicity of the alkoxyacetic acid metabolites of the glycol ethers.

Biokinetic modeling and in vitro-in vivo extrapolations.

The introduction of in vitro methodologies in the toxicological risk assessment process requires a number of prerequisites regarding both the toxicodynamics and the biokinetics of the compounds under study. In vitro systems will need to be relevant for measuring those structural and physiological changes that are good indicators for adverse effects. Furthermore, the dose metric found to have an effect in the in vitro system should be relevant. One element in defining the appropriate dose metric is related to the kinetic behavior of the compound in the in vitro system: binding to proteins, binding to plastic, evaporation, and the interaction between the culture medium and the cells. Ways to measure and model "in vitro biokinetics" are described. Second, the appropriate dose metric in vitro, e.g., the effective concentration, will need to be extrapolated to relevant in vivo exposure scenarios. The application of physiologically based biokinetic modelling is essential in such extrapolations. The parameters needed to build these models often can be estimated based on nonanimal data, namely chemical properties (QSARs) and in vitro experiments.

Human exposure to synthetic endocrine disrupting chemicals (S-EDCs) is generally negligible as compared to natural compounds with higher or comparable endocrine activity. How to evaluate the risk of the S-EDCs?

Theoretically, both synthetic endocrine disrupting chemicals (S-EDCs) and natural (exogenous and endogenous) endocrine disrupting chemicals (N-EDCs) can interact with endocrine receptors and disturb hormonal balance. However, compared to endogenous hormones, S-EDCs are only weak partial agonists with receptor affinities several orders of magnitude lower. Thus, to elicit observable effects, S-EDCs require considerably higher concentrations to attain sufficient receptor occupancy or to displace natural hormones and other endogenous ligands. Significant exposures to exogenous N-EDCs may result from ingestion of foods such as soy-based diets, green tea and sweet mustard. While their potencies are lower as compared to natural endogenous hormones, they usually are considerably more potent than S-EDCs. Effects of exogenous N-EDCs on the endocrine system were observed at high dietary intakes. A causal relation between their mechanism of action and these effects is established and biologically plausible. In contrast, the assumption that the much lower human exposures to S-EDCs may induce observable endocrine effects is not plausible. Hence, it is not surprising that epidemiological studies searching for an association between S-EDC exposure and health effects have failed. Regarding testing for potential endocrine effects, a scientifically justified screen should use in vitro tests to compare potencies of S-EDCs with those of reference N-EDCs. When the potency of the S-EDC is similar or smaller than that of the N-EDC, further testing in laboratory animals and regulatory consequences are not warranted.

Human exposure to synthetic endocrine disrupting chemicals (S-EDCs) is generally negligible as compared to natural compounds with higher or comparable endocrine activity. How to evaluate the risk of the S-EDCs?

Theoretically, both synthetic endocrine disrupting chemicals (S-EDCs) and natural (exogenous and endogenous) endocrine disrupting chemicals (N-EDCs) can interact with endocrine receptors and disturb hormonal balance. However, compared to endogenous hormones, S-EDCs are only weak partial agonists with receptor affinities several orders of magnitude lower. Thus, to elicit observable effects, S-EDCs require considerably higher concentrations to attain sufficient receptor occupancy or to displace natural hormones and other endogenous ligands. Significant exposures to exogenous N-EDCs may result from ingestion of foods such as soy-based diets, green tea and sweet mustard. While their potencies are lower as compared to natural endogenous hormones, they usually are considerably more potent than S-EDCs. Effects of exogenous N-EDCs on the endocrine system were observed at high dietary intakes. A causal relation between their mechanism of action and these effects is established and biologically plausible. In contrast, the assumption that the much lower human exposures to S-EDCs may induce observable endocrine effects is not plausible. Hence, it is not surprising that epidemiological studies searching for an association between S-EDC exposure and health effects have failed. Regarding testing for potential endocrine effects, a scientifically justified screen should use in vitro tests to compare potencies of S-EDCs with those of reference N-EDCs. When the potency of the S-EDC is similar or smaller than that of the N-EDC, further testing in laboratory animals and regulatory consequences are not warranted.

Human exposure to synthetic endocrine disrupting chemicals (S-EDCs) is generally negligible as compared to natural compounds with higher or comparable endocrine activity. How to evaluate the risk of the S-EDCs?

Theoretically, both synthetic endocrine disrupting chemicals (S-EDCs) and natural (exogenous and endogenous) endocrine disrupting chemicals (N-EDCs) can interact with endocrine receptors and disturb hormonal balance. However, compared to endogenous hormones, S-EDCs are only weak partial agonists with receptor affinities several orders of magnitude lower. Thus, to elicit observable effects, S-EDCs require considerably higher concentrations to attain sufficient receptor occupancy or to displace natural hormones and other endogenous ligands. Significant exposures to exogenous N-EDCs may result from ingestion of foods such as soy-based diets, green tea and sweet mustard. While their potencies are lower as compared to natural endogenous hormones, they usually are considerably more potent than S-EDCs. Effects of exogenous N-EDCs on the endocrine system were observed at high dietary intakes. A causal relation between their mechanism of action and these effects is established and biologically plausible. In contrast, the assumption that the much lower human exposures to S-EDCs may induce observable endocrine effects is not plausible. Hence, it is not surprising that epidemiological studies searching for an association between S-EDC exposure and health effects have failed. Regarding testing for potential endocrine effects, a scientifically justified screen should use in vitro tests to compare potencies of S-EDCs with those of reference N-EDCs. When the potency of the S-EDC is similar or smaller than that of the N-EDC, further testing in laboratory animals and regulatory consequences are not warranted.

Human exposure to synthetic endocrine disrupting chemicals (S-EDCs) is generally negligible as compared to natural compounds with higher or comparable endocrine activity. How to evaluate the risk of the S-EDCs?

Theoretically, both synthetic endocrine disrupting chemicals (S-EDCs) and natural (exogenous and endogenous) endocrine disrupting chemicals (N-EDCs) can interact with endocrine receptors and disturb hormonal balance. However, compared to endogenous hormones, S-EDCs are only weak partial agonists with receptor affinities several orders of magnitude lower. Thus, to elicit observable effects, S-EDCs require considerably higher concentrations to attain sufficient receptor occupancy or to displace natural hormones and other endogenous ligands. Significant exposures to exogenous N-EDCs may result from ingestion of foods such as soy-based diets, green tea and sweet mustard. While their potencies are lower as compared to natural endogenous hormones, they usually are considerably more potent. Effects of exogenous N-EDCs on the endocrine system were observed at high dietary intakes. A causal relation between their mechanism of action and these effects is established and biologically plausible. In contrast, the assumption that the much lower human exposures to S-EDCs may induce observable endocrine effects is not plausible. Hence, it is not surprising that epidemiological studies searching for an association between S-EDC exposure and health effects have failed. Regarding testing for potential endocrine effects, a scientifically justified screen should use in vitro tests to compare potencies of S-EDCs with those of reference N-EDCs. When the potency of the S-EDC is similar or smaller than that of the N-EDC, further testing in laboratory animals and regulatory consequences are not warranted.

New approach methodologies (NAMs) for human-relevant biokinetics predictions. Meeting the paradigm shift in toxicology towards an animal-free chemical risk assessment

For almost fifteen years, the availability and regulatory acceptance of new approach methodologies (NAMs) to assess the absorption, distribution, metabolism and excretion (ADME/biokinetics) in chemical risk evaluations are a bottleneck. To enhance the field, a team of 24 experts from science, industry, and regulatory bodies, including new generation toxicologists, met at the Lorentz Centre in Leiden, The Netherlands. A range of possibilities for the use of NAMs for biokinetics in risk evaluations were formulated (for example to define species differences and human variation or to perform quantitative in vitro-in vivo extrapolations). To increase the regulatory use and acceptance of NAMs for biokinetics for these ADME considerations within risk evaluations, the development of test guidelines (protocols) and of overarching guidance documents is considered a critical step. To this end, a need for an expert group on biokinetics within the Organisation of Economic Cooperation and Development (OECD) to supervise this process was formulated. The workshop discussions revealed that method development is still required, particularly to adequately capture transporter mediated processes as well as to obtain cell models that reflect the physiology and kinetic characteristics of relevant organs. Developments in the fields of stem cells, organoids and organ-on-a-chip models provide promising tools to meet these research needs in the future.

The contribution of in vitro toxicity data in hazard and risk assessment: current limitations and future perspectives.

This paper discusses the role of in vitro toxicology in hazard and risk assessment. A short historical overview of the development of the field of in vitro toxicology is given, showing the possibilities, as well as the limitation for using in vitro methods in screening and in studying mechanisms of toxicity. Although the development of the use of in vitro methods over the last two or three decades is vast, the application in risk assessment is still limited. The need for a more integrated approach, i.e. using in vitro data not in isolation, but in combination with data on structure-activity and in silico biokinetic models is discussed. It is foreseen that the role of in vitro methods in future risk assessments will greatly enhance, also in the light of recent developments in technologies such as genomics and systems biology.