Prediction of the dose range for adverse neurological effects of amiodarone in patients from an in vitro toxicity test by in vitro-in vivo extrapolation.

Amiodarone is an antiarrhythmic agent inducing adverse effects on the nervous system, among others. We applied physiologically based pharmacokinetic (PBPK) modeling combined with benchmark dose modeling to predict, based on published in vitro data, the in vivo dose of amiodarone which may lead to adverse neurological effects in patients. We performed in vitro-in vivo extrapolation (IVIVE) from concentrations measured in the cell lysate of a rat brain 3D cell model using a validated human PBPK model. Among the observed in vitro effects, inhibition of choline acetyl transferase (ChAT) was selected as a marker for neurotoxicity. By reverse dosimetry, we transformed the in vitro concentration-effect relationship into in vivo effective human doses, using the calculated in vitro area under the curve (AUC) of amiodarone as the pharmacokinetic metric. The upper benchmark dose (BMDU) was calculated and compared with clinical doses eliciting neurological adverse effects in patients. The AUCs in the in vitro brain cell culture after 14-day repeated dosing of nominal concentration equal to 1.25 and 2.5 µM amiodarone were 1.00 and 1.99 µg*h/mL, respectively. The BMDU was 385.4 mg for intravenous converted to 593 mg for oral application using the bioavailability factor of 0.65 as reported in the literature. The predicted dose compares well with neurotoxic doses in patients supporting the hypothesis that impaired ChAT activity may be related to the molecular/cellular mechanisms of amiodarone neurotoxicity. Our study shows that predicting effects from in vitro data together with IVIVE can be used at the initial stage for the evaluation of potential adverse drug reactions and safety assessment in humans.

The EU chemicals strategy for sustainability: in support of the BfR position.

The EU chemicals strategy for sustainability (CSS) asserts that both human health and the environment are presently threatened and that further regulation is necessary. In a recent Guest Editorial, members of the German competent authority for risk assessment, the BfR, raised concerns about the scientific justification for this strategy. The complexity and interdependence of the networks of regulation of chemical substances have ensured that public health and wellbeing in the EU have continuously improved. A continuous process of improvement in consumer protection is clearly desirable but any initiative directed towards this objective must be based on scientific knowledge. It must not confound risk with other factors in determining policy. This conclusion is fully supported in the present Commentary including the request to improve both, data collection and the time-consuming and bureaucratic procedures that delay the publication of regulations.

Advice to the European Commission as Regards Type and Criteria for Comprehensive Studies to Be Requested From Manufacturers: The Opinion of the Scientific Committee on Health, Environmental, and Emerging Risks (SCHEER).

The European Commission has established a priority list of 15 additives contained in cigarettes and roll-your-own tobacco subject to enhanced reporting obligations. The European Union (EU) Tobacco Products Directive (TPD) prescribes that Member States shall require manufacturers and importers of tobacco products to carry out comprehensive studies on these additives to assess their contribution to any of the properties listed in Article 6 of the TPD: toxicity or addictiveness, characterizing flavor, inhalation facilitation, nicotine uptake, and carcinogenic, mutagenic, or toxic for reproduction. The Scientific Committee on Health, Environmental, and Emerging Risks (SCHEER) has provided guidance on the type and criteria for comprehensive studies, and on the most suitable methodologies to test these 15 tobacco additives as well as additives on future updated lists. The SCHEER proposes a stepwise strategy as the most pragmatic and efficient way to assess the effects of tobacco additives. In addition to proposing specific steps and tests to be considered by industry, some general criteria were also identified such as no comparative testing (testing cigarettes with and without the additive) and no animal studies. As tobacco additives have no benefits for health, but rather may promote use of and addiction to an extremely toxic product, a risk-benefit analysis is not the appropriate paradigm for assessing the additive. When comprehensive studies confirm that additives have any of the properties listed in Article 6 of the TPD, regulatory actions should be considered. If uncertainties cannot be solved by comprehensive studies, the SCHEER recommends that the assessors consider the worst-case evaluation. IMPLICATIONS: In this article, the SCHEER proposes a stepwise strategy to assess (1) the toxic and addictive effects, (2) the characterizing flavor, and (3) facilitating inhalation properties of tobacco additives. The proposed steps and tests provide guidance to (1) Member State on which comprehensive studies should be requested and (2) tobacco industry on which strategy of testing should be applied to address the request and to prepare reports to be sent to the relevant authorities for the evaluation of tobacco additives "safety" to comply with the Tobacco Products Directive 2014/40/EU.

Cyanotoxins: producing organisms, occurrence, toxicity, mechanism of action and human health toxicological risk evaluation.

Cyanobacteria were present on the earth 3.5 billion years ago; since then they have colonized almost all terrestrial and aquatic ecosystems. They produce a high number of bioactive molecules, among which some are cyanotoxins. Cyanobacterial growth at high densities, forming blooms, is increasing in extension and frequency, following anthropogenic activities and climate changes, giving rise to some concern for human health and animal life exposed to cyanotoxins. Numerous cases of lethal poisonings have been associated with cyanotoxins ingestion in wild animal and livestock. In humans few episodes of lethal or severe human poisonings have been recorded after acute or short-term exposure, but the repeated/chronic exposure to low cyanotoxin levels remains a critical issue. The properties of the most frequently detected cyanotoxins (namely, microcystins, nodularins, cylindrospermopsin and neurotoxins) are here critically reviewed, describing for each toxin the available information on producing organisms, biosynthesis/genetic and occurrence, with a focus on the toxicological profile (including kinetics, acute systemic toxicity, mechanism and mode of action, local effects, repeated toxicity, genotoxicity, carcinogenicity, reproductive toxicity; human health effects and epidemiological studies; animal poisoning) with the derivation of health-based values and considerations on the risks for human health.

Risk to human health associated with the environmental occurrence of cyanobacterial neurotoxic alkaloids anatoxins and saxitoxins.

Cyanobacteria are ubiquitous photosynthetic micro-organisms forming blooms and scums in surface water; among them some species can produce cyanotoxins giving rise to some concern for human health and animal life. To date, more than 65 cyanobacterial neurotoxins have been described, of which the most studied are the groups of anatoxins and saxitoxins (STXs), comprising many different variants. In freshwaters, the hepatotoxic microcystins represent the most frequently detected cyanotoxin: on this basis, it could appear that neurotoxins are less relevant, but the low frequency of detection may partially reflect an a priori choice of target analytes, the low method sensitivity and the lack of certified standards. Cyanobacterial neurotoxins target cholinergic synapses or voltage-gated ion channels, blocking skeletal and respiratory muscles, thus leading to death by respiratory failure. This review reports and analyzes the available literature data on environmental occurrence of cyanobacterial neurotoxic alkaloids, namely anatoxins and STXs, their biosynthesis, toxicology and epidemiology, derivation of guidance values and action limits. These data are used as the basis to assess the risk posed to human health, identify critical exposure scenarios and highlight the major data gaps and research needs.

Metals in cosmetics: an a posteriori safety evaluation.

According to EU Regulation No. 1223/2009/CE cosmetic products for daily use can contain 'technically unavoidable traces' of metals. This definition is too vague. Authorities should set well-defined limits, considering the risks associated with metal contamination of personal care products (PCPs). This paper characterizes the risk arising from a number of metals (antimony, arsenic, cadmium, cobalt, chromium, mercury, nickel, lead) that may occur in 'unavoidable traces" in raw materials and, consequently, in PCPs. A 'worst case scenario' was adopted, based on the following assumptions: (i) the individual ingredients contained the maximum amount in traces allowed for each metal; (ii) the hypothetical PCP was produced exclusively with that single ingredient; (iii) when absorption through the skin was not known, data related to oral absorption were used. Risk characterization was performed calculating the Systemic Exposure Dosage (SED) and the Margin of Safety (MoS=NOAEL or BMDL10/SED). Exposure to the allegedly 'technically unavoidable' maximum amounts of metals in cosmetic ingredients resulted in MoSs exceeding 100 (safety threshold) with one exception. This suggests that the availability of experimental dermal absorption rates could enable significant improvement in MoS, thus increasing safety levels. Although results are reassuring, the authors recommend minimization of contamination, according to the state of the art of manufacturing methods.

PBTK modelling platforms and parameter estimation tools to enable animal-free risk assessment: recommendations from a joint EPAA--EURL ECVAM ADME workshop.

Information on toxicokinetics is critical for animal-free human risk assessment. Human external exposure must be translated into human tissue doses and compared with in vitro actual cell exposure associated to effects (in vitro-in vivo comparison). Data on absorption, distribution, metabolism and excretion in humans (ADME) could be generated using in vitro and QSAR tools. Physiologically-based toxicokinetic (PBTK) computer modelling could serve to integrate disparate in vitro and in silico findings. However, there are only few freely-available PBTK platforms currently available. And although some ADME parameters can be reasonably estimated in vitro or in silico, important gaps exist. Examples include unknown or limited applicability domains and lack of (high-throughput) tools to measure penetration of barriers, partitioning between blood and tissues and metabolic clearance. This paper is based on a joint EPAA--EURL ECVAM expert meeting. It provides a state-of-the-art overview of the availability of PBTK platforms as well as the in vitro and in silico methods to parameterise basic (Tier 1) PBTK models. Five high-priority issues are presented that provide the prerequisites for wider use of non-animal based PBTK modelling for animal-free chemical risk assessment.

Co-Occurrence of Taste and Odor Compounds and Cyanotoxins in Cyanobacterial Blooms: Emerging Risks to Human Health?

Cyanobacteria commonly form large blooms in waterbodies; they can produce cyanotoxins, with toxic effects on humans and animals, and volatile compounds, causing bad tastes and odors (T&O) at naturally occurring low concentrations. Notwithstanding the large amount of literature on either cyanotoxins or T&O, no review has focused on them at the same time. The present review critically evaluates the recent literature on cyanotoxins and T&O compounds (geosmin, 2-methylisoborneol, ß-ionone and ß-cyclocitral) to identify research gaps on harmful exposure of humans and animals to both metabolite classes. T&O and cyanotoxins production can be due to the same or common to different cyanobacterial species/strains, with the additional possibility of T&O production by non-cyanobacterial species. The few environmental studies on the co-occurrence of these two groups of metabolites are not sufficient to understand if and how they can co-vary, or influence each other, perhaps stimulating cyanotoxin production. Therefore, T&Os cannot reliably serve as early warning surrogates for cyanotoxins. The scarce data on T&O toxicity seem to indicate a low health risk (but the inhalation of ß-cyclocitral deserves more study). However, no data are available on the effects of combined exposure to mixtures of cyanotoxins and T&O compounds and to combinations of T&O compounds; therefore, whether the co-occurrence of cyanotoxins and T&O compounds is a health issue remains an open question.

Prediction of in vivo prenatal chlorpyrifos exposure leading to developmental neurotoxicity in humans based on in vitro toxicity data by quantitative in vitro-in vivo extrapolation.

Introduction: Epidemiological studies in children suggested that in utero exposure to chlorpyrifos (CPF), an organophosphate insecticide, may cause developmental neurotoxicity (DNT). We applied quantitative in vitro-in vivo extrapolation (QIVIVE) based on in vitro concentration and non-choline esterase-dependent effects data combined with Benchmark dose (BMD) modelling to predict oral maternal CPF exposure during pregnancy leading to fetal brain effect concentration. By comparing the results with data from epidemiological studies, we evaluated the contribution of the in vitro endpoints to the mode of action (MoA) for CPF-induced DNT. Methods: A maternal-fetal PBK model built in PK-Sim® was used to perform QIVIVE predicting CPF concentrations in a pregnant women population at 15 weeks of gestation from cell lysate concentrations obtained in human induced pluripotent stem cell-derived neural stem cells undergoing differentiation towards neurons and glia exposed to CPF for 14 days. The in vitro concentration and effect data were used to perform BMD modelling. Results: The upper BMD was converted into maternal doses which ranged from 3.21 to 271 mg/kg bw/day. Maternal CPF blood levels from epidemiological studies reporting DNT findings in their children were used to estimate oral CPF exposure during pregnancy using the PBK model. It ranged from 0.11 to 140 µg/kg bw/day. Discussion: The effective daily intake doses predicted from the in vitro model were several orders of magnitude higher than exposures estimated from epidemiological studies to induce developmental non-cholinergic neurotoxic responses, which were captured by the analyzed in vitro test battery. These were also higher than the in vivo LOEC for cholinergic effects. Therefore, the quantitative predictive value of the investigated non-choline esterase-dependent effects, although possibly relevant for other chemicals, may not adequately represent potential key events in the MoA for CPF-associated DNT.

Metabolites in the regulatory risk assessment of pesticides in the EU.

A large majority of chemicals is converted into metabolites through xenobiotic-metabolising enzymes. Metabolites may present a spectrum of characteristics varying from similar to vastly different compared with the parent compound in terms of both toxicokinetics and toxicodynamics. In the pesticide arena, the role of metabolism and metabolites is increasingly recognised as a significant factor particularly for the design and interpretation of mammalian toxicological studies and in the toxicity assessment of pesticide/metabolite-associated issues for hazard characterization and risk assessment purposes, including the role of metabolites as parts in various residues in ecotoxicological adversities. This is of particular relevance to pesticide metabolites that are unique to humans in comparison with metabolites found in in vitro or in vivo animal studies, but also to disproportionate metabolites (quantitative differences) between humans and mammalian species. Presence of unique or disproportionate metabolites may underlie potential toxicological concerns. This review aims to present the current state-of-the-art of comparative metabolism and metabolites in pesticide research for hazard and risk assessment, including One Health perspectives, and future research needs based on the experiences gained at the European Food Safety Authority.

Protocol for designing INVITES-IN, a tool for assessing the internal validity of in vitro studies.

This protocol describes the design and development of a tool for evaluation of the internal validity of in vitro studies, which is needed to include the data as evidence in systematic reviews and chemical risk assessments. The tool will be designed specifically to be applied to cell culture studies, including, but not restricted to, studies meeting the new approach methodology (NAM) definition. The tool is called INVITES-IN (IN VITro Experimental Studies INternal validity). In this protocol, three of the four studies that will be performed to create the release version of INVITES-IN are described. In the first study, evaluation of existing assessment tools will be combined with focus group discussions to identify how characteristics of the design or conduct of an in vitro study can affect its internal validity. Bias domains and items considered to be of relevance for in vitro studies will be identified. In the second study, group agreement on internal validity domains and items of importance for in vitro studies will be identified via a modified Delphi methodology. In the third study, the draft version of the tool will be created, based on the data on relevance and importance of bias domains and items collected in Studies 1 and 2. A separate protocol will be prepared for the fourth study, which includes the user testing and validation of the tool, and collection of users' experience.

Congeners-Specific Intestinal Absorption Of Microcystins In An In Vitro 3D Human Intestinal Epithelium: The Role Of Influx/Efflux Transporters.

Microcystins constitute a group of over 200 variants and are increasingly considered as emerging toxins in food and feed safety, particularly with regards to sea-food and fish consumption. Toxicity of MCs is congener-specific, being characterised by different acute potencies, likely related to the differential activity of metabolic enzymes and transporters proteins involved in their cellular uptake. However, the active transport of MCs across intestinal membranes has not been fully elucidated. Our results, obtained using a fit for purpose 3D human reconstructed intestinal epithelium, provide new information on the complex mechanisms involved in the absorption of 5 MC variants': it is indeed characterised by the equilibrium between uptake and extrusion, since the selected congeners are substrates of both influx and efflux proteins. In the range of tested nominal concentrations (10-40 µM) fully representative of relevant exposure scenarios, none of the active tested transporters were saturated. The comparison of permeability (Papp) values of MCs variants highlighted a dose independent relationship for MC-LR, -YR and -RR (Papp x 10-7 ranged from 2.95 to 3.54 cm/s), whereas -LW and-LF showed a dose dependent increase in permeability reaching Papp values which were similar to the other congeners at 40 µM. MC-RR, -LR, -YR show absorption values around 5% of the administered dose. Due to their lipophilicity, MC-LW and -LF were also detected within the cellular compartment. The intestinal uptake was only partially attributable to OATPs, suggesting the involvement of additional transporters. Regarding the efflux proteins, MCs are not P-gp substrates whereas MRP2 and to a lesser extent Breast cancer resistance protein are active in their extrusion. Despite the presence of GST proteins, as an indication of metabolic competence, in the intestinal tissue, MC-conjugates were never detected in our experimental settings.

In Vitro-In Vivo Extrapolation by Physiologically Based Kinetic Modeling: Experience With Three Case Studies and Lessons Learned.

Physiologically based kinetic (PBK) modeling has been increasingly used since the beginning of the 21st century to support dose selection to be used in preclinical and clinical safety studies in the pharmaceutical sector. For chemical safety assessment, the use of PBK has also found interest, however, to a smaller extent, although an internationally agreed document was published already in 2010 (IPCS/WHO), but at that time, PBK modeling was based mostly on in vivo data as the example in the IPCS/WHO document indicates. Recently, the OECD has published a guidance document which set standards on how to characterize, validate, and report PBK models for regulatory purposes. In the past few years, we gained experience on using in vitro data for performing quantitative in vitro-in vivo extrapolation (QIVIVE), in which biokinetic data play a crucial role to obtain a realistic estimation of human exposure. In addition, pharmaco-/toxicodynamic aspects have been introduced into the approach. Here, three examples with different drugs/chemicals are described, in which different approaches have been applied. The lessons we learned from the exercise are as follows: 1) in vitro conditions should be considered and compared to the in vivo situation, particularly for protein binding; 2) in vitro inhibition of metabolizing enzymes by the formed metabolites should be taken into consideration; and 3) it is important to extrapolate from the in vitro measured intracellular concentration and not from the nominal concentration to the tissue/organ concentration to come up with an appropriate QIVIVE for the relevant adverse effects.

Human glutathione transferases catalyzing the conjugation of the hepatoxin microcystin-LR.

Many cyanobacterial species are able to produce cyanotoxins as secondary metabolites. Among them, microcystins (MC) are a group of around 80 congeners of toxic cyclic heptapeptides. MC-LR is the most studied MC congener, in view of its high acute hepatotoxicity and tumor promoting activity. Humans may be exposed to cyanotoxins through several routes, the oral one being the most important. The accepted pathway for MC-LR detoxication and excretion in the urine is GSH conjugation. The GSH adduct (GS-MCLR) formation has been shown to occur spontaneously and enzymatically, catalyzed by glutathione transferases (GSTs). The enzymatic reaction has been reported but not characterized both in vitro and in vivo in animal and plant species. No data are available on humans. In the present work, the MC-LR conjugation with GSH catalyzed by five recombinant human GSTs (A1-1, A3-3, M1-1, P1-1, and T1-1) has been characterized for the first time. All GSTs are able to catalyze the reaction; kinetic parameters K(m), k(cat), and their relative specific activities to form GS-MCLR were derived (T1-1 > A1-1 > M1-1 > A3-3 ≫ P1-1). In the range of MC tested concentrations used (0.25-50 µM) GSTT1-1 and A1-1 showed a typical saturation curve with similar affinity for MC-LR (≈80 µM; k(cat) values 0.18 and 0.10 min(-1), respectively), A3-3 and M1-1 were linear, whereas GSTP1-1 showed a temperature-dependent sigmoidal allosteric curve with a k(cat) = 0.11 min(-1). The enzymes mainly expressed in the liver and gastrointestinal tract, GSTA1-1, T1-1, and M1-1, seemed to be mainly involved in the MC-LR detoxification after oral exposure, whereas P1-1 kinetics and location in the skin suggest a role related to dermal exposure. Considering the high frequency of some GST polymorphism, especially M1 and T1 gene deletion, with complete loss in activity, this information could be the first step to identify groups of individual at higher risk associated with MC exposure.

Alternative (non-animal) methods for cosmetics testing: current status and future prospects-2010.

The 7th amendment to the EU Cosmetics Directive prohibits to put animal-tested cosmetics on the market in Europe after 2013. In that context, the European Commission invited stakeholder bodies (industry, non-governmental organisations, EU Member States, and the Commission's Scientific Committee on Consumer Safety) to identify scientific experts in five toxicological areas, i.e. toxicokinetics, repeated dose toxicity, carcinogenicity, skin sensitisation, and reproductive toxicity for which the Directive foresees that the 2013 deadline could be further extended in case alternative and validated methods would not be available in time. The selected experts were asked to analyse the status and prospects of alternative methods and to provide a scientifically sound estimate of the time necessary to achieve full replacement of animal testing. In summary, the experts confirmed that it will take at least another 7-9 years for the replacement of the current in vivo animal tests used for the safety assessment of cosmetic ingredients for skin sensitisation. However, the experts were also of the opinion that alternative methods may be able to give hazard information, i.e. to differentiate between sensitisers and non-sensitisers, ahead of 2017. This would, however, not provide the complete picture of what is a safe exposure because the relative potency of a sensitiser would not be known. For toxicokinetics, the timeframe was 5-7 years to develop the models still lacking to predict lung absorption and renal/biliary excretion, and even longer to integrate the methods to fully replace the animal toxicokinetic models. For the systemic toxicological endpoints of repeated dose toxicity, carcinogenicity and reproductive toxicity, the time horizon for full replacement could not be estimated.

OpenCYP: An open source database exploring human variability in activities and frequencies of polymophisms for major cytochrome P-450 isoforms across world populations.

The open source database "OpenCYP database" has been developed based on the results of extensive literature searches from the peer-reviewed literature. OpenCYP provides data on human variability on baseline of activities and polymophism frequencies for selected cytochrome P-450 isoforms (CYP1A2, CYP2A6, CYP2D6, CYP3A4/3A5 and CYP3A7) in healthy adult populations from world populations. CYP enzymatic activities were generally expressed as the metabolic ratio (MR) between an unchanged probe drug and its metabolite(s) in urine or plasma measured in healthy adults. Data on other age groups were very limited and fragmented, constituting an important data gap. Quantitative comparisons were often hampered by the different experimental conditions used. However, variability was quite limited for CYP1A2, using caffeine as a probe substrate, with a symmetrical distribution of metabolic activity values. For CYP3A4, human variability was dependent on the probe substrate itself with low variability when data considering the dextromethorphan/demethilathed metabolite MR were used and large variability when the urinary 6ß-hydroxycortisol/cortisol ratio was used. The largest variability in CYP activity was shown for CYP2D6 activity, after oral dosing of dextromethorphan, for which genetic polymorphisms are well characterised and constitute a significant source of variability. It is foreseen that the OpenCYP database can contribute to promising tools to support the further development of QIVIVE and PBK models for human risk assessment of chemicals particularly when combined with information on isoform-specific content in cells using proteomic approaches.