Comparative case study on NAMs: towards enhancing specific target organ toxicity analysis.

Traditional risk assessment methodologies in toxicology have relied upon animal testing, despite concerns regarding interspecies consistency, reproducibility, costs, and ethics. New Approach Methodologies (NAMs), including cell culture and multi-level omics analyses, hold promise by providing mechanistic information rather than assessing organ pathology. However, NAMs face limitations, like lacking a whole organism and restricted toxicokinetic interactions. This is an inherent challenge when it comes to the use of omics data from in vitro studies for the prediction of organ toxicity in vivo. One solution in this context are comparative in vitro-in vivo studies as they allow for a more detailed assessment of the transferability of the respective NAM data. Hence, hepatotoxic and nephrotoxic pesticide active substances were tested in human cell lines and the results subsequently related to the biology underlying established effects in vivo. To this end, substances were tested in HepaRG and RPTEC/tERT1 cells at non-cytotoxic concentrations and analyzed for effects on the transcriptome and parts of the proteome using quantitative real-time PCR arrays and multiplexed microsphere-based sandwich immunoassays, respectively. Transcriptomics data were analyzed using three bioinformatics tools. Where possible, in vitro endpoints were connected to in vivo observations. Targeted protein analysis revealed various affected pathways, with generally fewer effects present in RPTEC/tERT1. The strongest transcriptional impact was observed for Chlorotoluron in HepaRG cells (increased CYP1A1 and CYP1A2 expression). A comprehensive comparison of early cellular responses with data from in vivo studies revealed that transcriptomics outperformed targeted protein analysis, correctly predicting up to 50% of in vivo effects.

Thresholds of adversity for endocrine disrupting substances: a conceptual case study.

For endocrine disrupting chemicals (EDC) the existence of "safe exposure levels", that is exposure levels that do not present an appreciable risk to human health is most controversially discussed, as is the existence of health-based reference values. Concerns have been especially raised that EDCs might not possess a threshold level such that no exposure level to EDCs can be considered safe. To explore whether or not threshold levels can be identified, we performed a screening exercise on 14 pesticidal and biocidal active substances previously identified as EDCs in the European Union. The respective substances are ideal subjects for case studies to review for endocrine activity and disruptive potential following well-defined regulatory assessment based on solid data to effectually establish adversity as consequence of endocrine disruption. Dimethomorph, metiram and propiconazole for which the weight of evidence demonstrating endocrine disruption was the strongest were used as subjects for further study. Epoxiconazole was additionally selected as its effects on the endocrine system are extensive. For all four substances, analysis of the toxicological data clearly indicated thresholds of adversity below which no adverse effects mediated through an endocrine mechanism were observed. Particular emphasis was placed on mechanistic considerations including homeostasis and the concept of adversity. As a proof of concept this study provides evidence that like other substances of toxicological concern EDCs have threshold levels for adversity. While for some EDCs the respective thresholds might indeed be very low this shows that, data allowing, for other EDCs sufficiently protective reference values can be derived.

Genotoxicity assessment: opportunities, challenges and perspectives for quantitative evaluations of dose-response data.

Genotoxicity data are mainly interpreted in a qualitative way, which typically results in a binary classification of chemical entities. For more than a decade, there has been a discussion about the need for a paradigm shift in this regard. Here, we review current opportunities, challenges and perspectives for a more quantitative approach to genotoxicity assessment. Currently discussed opportunities mainly include the determination of a reference point (e.g., a benchmark dose) from genetic toxicity dose-response data, followed by calculation of a margin of exposure (MOE) or derivation of a health-based guidance value (HBGV). In addition to new opportunities, major challenges emerge with the quantitative interpretation of genotoxicity data. These are mainly rooted in the limited capability of standard in vivo genotoxicity testing methods to detect different types of genetic damage in multiple target tissues and the unknown quantitative relationships between measurable genotoxic effects and the probability of experiencing an adverse health outcome. In addition, with respect to DNA-reactive mutagens, the question arises whether the widely accepted assumption of a non-threshold dose-response relationship is at all compatible with the derivation of a HBGV. Therefore, at present, any quantitative genotoxicity assessment approach remains to be evaluated case-by-case. The quantitative interpretation of in vivo genotoxicity data for prioritization purposes, e.g., in connection with the MOE approach, could be seen as a promising opportunity for routine application. However, additional research is needed to assess whether it is possible to define a genotoxicity-derived MOE that can be considered indicative of a low level of concern. To further advance quantitative genotoxicity assessment, priority should be given to the development of new experimental methods to provide a deeper mechanistic understanding and a more comprehensive basis for the analysis of dose-response relationships.

Basic concepts of mixture toxicity and relevance for risk evaluation and regulation.

Exposure to multiple substances is a challenge for risk evaluation. Currently, there is an ongoing debate if generic "mixture assessment/allocation factors" (MAF) should be introduced to increase public health protection. Here, we explore concepts of mixture toxicity and the potential influence of mixture regulation concepts for human health protection. Based on this analysis, we provide recommendations for research and risk assessment. One of the concepts of mixture toxicity is additivity. Substances may act additively by affecting the same molecular mechanism within a common target cell, for example, dioxin-like substances. In a second concept, an "enhancer substance" may act by increasing the target site concentration and aggravating the adverse effect of a "driver substance". For both concepts, adequate risk management of individual substances can reliably prevent adverse effects to humans. Furthermore, we discuss the hypothesis that the large number of substances to which humans are exposed at very low and individually safe doses may interact to cause adverse effects. This commentary identifies knowledge gaps, such as the lack of a comprehensive overview of substances regulated under different silos, including food, environmentally and occupationally relevant substances, the absence of reliable human exposure data and the missing accessibility of ratios of current human exposure to threshold values, which are considered safe for individual substances. Moreover, a comprehensive overview of the molecular mechanisms and most susceptible target cells is required. We conclude that, currently, there is no scientific evidence supporting the need for a generic MAF. Rather, we recommend taking more specific measures, which focus on compounds with relatively small ratios between human exposure and doses, at which adverse effects can be expected.

Applying genomics in regulatory toxicology: a report of the ECETOC workshop on omics threshold on non-adversity.

In a joint effort involving scientists from academia, industry and regulatory agencies, ECETOC's activities in Omics have led to conceptual proposals for: (1) A framework that assures data quality for reporting and inclusion of Omics data in regulatory assessments; and (2) an approach to robustly quantify these data, prior to interpretation for regulatory use. In continuation of these activities this workshop explored and identified areas of need to facilitate robust interpretation of such data in the context of deriving points of departure (POD) for risk assessment and determining an adverse change from normal variation. ECETOC was amongst the first to systematically explore the application of Omics methods, now incorporated into the group of methods known as New Approach Methodologies (NAMs), to regulatory toxicology. This support has been in the form of both projects (primarily with CEFIC/LRI) and workshops. Outputs have led to projects included in the workplan of the Extended Advisory Group on Molecular Screening and Toxicogenomics (EAGMST) group of the Organisation for Economic Co-operation and Development (OECD) and to the drafting of OECD Guidance Documents for Omics data reporting, with potentially more to follow on data transformation and interpretation. The current workshop was the last in a series of technical methods development workshops, with a sub-focus on the derivation of a POD from Omics data. Workshop presentations demonstrated that Omics data developed within robust frameworks for both scientific data generation and analysis can be used to derive a POD. The issue of noise in the data was discussed as an important consideration for identifying robust Omics changes and deriving a POD. Such variability or "noise" can comprise technical or biological variation within a dataset and should clearly be distinguished from homeostatic responses. Adverse outcome pathways (AOPs) were considered a useful framework on which to assemble Omics methods, and a number of case examples were presented in illustration of this point. What is apparent is that high dimension data will always be subject to varying processing pipelines and hence interpretation, depending on the context they are used in. Yet, they can provide valuable input for regulatory toxicology, with the pre-condition being robust methods for the collection and processing of data together with a comprehensive description how the data were interpreted, and conclusions reached.

Toxicokinetic and toxicodynamic mixture effects of plant protection products: A case study.

Authorisation of ready to use plant protection products (PPPs) usually relies on the testing of acute and local toxicity only. This is in stark contrast to the situation for active substances where the mandatory data set comprises a most comprehensive set of studies. While the combination of certain active ingredients and co-formulants may nevertheless result in increased toxicity of the final product such combinations have never been evaluated systematically for complex and long-term toxicological endpoints. We therefore investigated the effect of three frequently used co-formulants on the toxicokinetic and toxicodynamic of the representative active substance combination of tebuconazol (Teb) and prothioconazol (Pro) or of cypermethrin (Cpm) and piperonyl butoxide (Pip), respectively. With all four active substances being potential liver steatogens, cytotoxicity and triglyceride accumulation in HepaRG were used as primary endpoints. Concomitantly transcriptomics and biochemical studies were applied to interrogate for effects on gene expression or inhibition of CYP3A4 as key enzyme for functionalization. Some of the tested combinations clearly showed more than additive effects, partly due to CYP3A4 enzyme inhibition. Other effects comprised the modulation of the expression and activity of steatosis-related nuclear key receptors. Altogether, the findings highlight the need for a more systematic consideration of toxicodynamic and toxicokinetic mixture effects during assessment of PPPs.

An oxidative N-demethylase reveals PAS transition from ubiquitous sensor to enzyme.

The universal Per-ARNT-Sim (PAS) domain functions as a signal transduction module involved in sensing diverse stimuli such as small molecules, light, redox state and gases. The highly evolvable PAS scaffold can bind a broad range of ligands, including haem, flavins and metal ions. However, although these ligands can support catalytic activity, to our knowledge no enzymatic PAS domain has been found. Here we report characterization of the first PAS enzyme: a haem-dependent oxidative N-demethylase. Unrelated to other amine oxidases, this enzyme contains haem, flavin mononucleotide, 2Fe-2S and tetrahydrofolic acid cofactors, and specifically catalyses the NADPH-dependent oxidation of dimethylamine. The structure of the α subunit reveals that it is a haem-binding PAS domain, similar in structure to PAS gas sensors. The dimethylamine substrate forms part of a highly polarized oxygen-binding site, and directly assists oxygen activation by acting as both an electron and proton donor. Our data reveal that the ubiquitous PAS domain can make the transition from sensor to enzyme, suggesting that the PAS scaffold can support the development of artificial enzymes.

An approach for mixture testing and prioritization based on common kinetic groups.

In light of an ever-increasing exposure to chemicals, the topic of potential mixture toxicity has gained increased attention, particularly as the toxicological toolbox to address such questions has vastly improved. Routinely toxicological risk assessments will rely on the analysis of individual compounds with mixture effects being considered only in those specific cases where co-exposure is foreseeable, for example for pesticides or food contact materials. In the field of pesticides, active substances are summarized in so-called cumulative assessment groups (CAG) which are primarily based on their toxicodynamic properties, that is, respective target organs and mode of action (MoA). In this context, compounds causing toxicity by a similar MoA are assumed to follow a model of dose/concentration addition (DACA). However, the respective approach inherently falls short of addressing cases where there are dissimilar or independent MoAs resulting in wider toxicokinetic effects. Yet, the latter are often the underlying cause when effects deviate from the DACA model. In the present manuscript, we therefore suggest additionally to consider toxicokinetic effects (especially related to xenobiotic metabolism and transporter interaction) for the grouping of substances to predict mixture toxicity. In line with the concept of MoA-based CAGs, we propose common kinetics groups (CKGs) as an additional tool for grouping of chemicals and mixture prioritization. Fundamentals of the CKG concept are discussed, along with challenges for its implementation, and methodological approaches and examples are explored.

Effects of co-formulants on the absorption and secretion of active substances in plant protection products in vitro.

Currently, the authorisation process for plant protection products (PPPs) relies on the testing of acute and topological toxicity only. Contrastingly, the evaluation of active substances includes a more comprehensive set of toxicity studies. Nevertheless, mixture effects of active ingredients and co-formulants may result in increased toxicity. Therefore, we investigated effects of surface active co-formulants on the toxicity of two PPPs focussing on qualitative and quantitative toxicokinetic effects on absorption and secretion. The respective products are based on the active substances abamectin and fluroxypyr-meptyl and were tested for cytotoxicity in the presence or absence of the corresponding surfactants and co-formulants using Caco-2 cells. In addition, the effect of co-formulants on increased cellular permeation was quantified using LC-MS/MS, while potential kinetic mixture effects were addressed by fluorescence anisotropy measurements and ATPase assays. The results show that surface active co-formulants significantly increase the cytotoxicity of the investigated PPPs, leading to more than additive mixture effects. Moreover, analytical investigations show higher efflux ratios of both active substances and the metabolite fluroxypyr upon combination with certain concentrations of the surfactants. The results further point to a significant and concentration-dependent inhibition of Pgp transporters by most of the surfactants as well as to increased membrane fluidity. Altogether, these findings strongly support the hypothesis that surfactants contribute to increased cytotoxicity of PPPs and do so by increasing the bioavailability of the respective active substances.

Characterization of Quinoline Yellow Dyes As Transient Aryl Hydrocarbon Receptor Agonists.

The aryl hydrocarbon receptor (AHR) and estrogen receptor alpha (ERα) are two ligand activated transcription factors that are targeted by a wide range of anthropogenic compounds. Crosstalk between both receptors is well established but little understood. We previously developed a dual color luciferase assay (i.e., XEER) which allows time dissolved monitoring of the activation of both receptors in situ. The system was now used in conjunction with HPLC-qTOF to identify several quinophthalone dyes as transient receptor agonists of the AHR. Altogether the approach identified three widely used dyes, that is the plastic colorant latyl yellow 3G (LY), the structurally related textile dye disperse yellow 64 (DY), and the cosmetic dye quinoline yellow (QY). The latter was the most potent agonist followed by LY and DY as confirmed by the XEER assay and CYP1A1 gene induction in MCF7 cells. In addition QY, LY, and DY also inhibited ER signaling in an AHR-dependent manner. This establishes some evidence for quinoline yellow dyes as potential disruptors of AHR/ER signaling, raising potential toxicological concern. Although none of the dyes featured any signs of genotoxicity in vitro, our data point to the need for a systematic approach when screening for substances of potential toxicological and endocrine relevance.

Microbially competent 3D skin: a test system that reveals insight into host-microbe interactions and their potential toxicological impact.

The skin`s microbiome is predominantly commensalic, harbouring a metabolic potential far exceeding that of its host. While there is clear evidence that bacteria-dependent metabolism of pollutants modulates the toxicity for the host there is still a lack of models for investigating causality of microbiome-associated pathophysiology or toxicity. We now report on a biologically characterised microbial-skin tissue co-culture that allows studying microbe-host interactions for extended periods of time in situ. The system is based on a commercially available 3D skin model. In a proof-of-concept, this model was colonised with single and mixed cultures of two selected skin commensals. Two different methods were used to quantify the bacteria on the surface of the skin models. While Micrococcus luteus established a stable microbial-skin tissue co-culture, Pseudomonas oleovorans maintained slow continuous growth over the 8-day cultivation period. A detailed skin transcriptome analysis showed bacterial colonisation leading to up to 3318 significant changes. Additionally, FACS, ELISA and Western blot analyses were carried out to analyse secretion of cytokines and growth factors. Changes found in colonised skin varied depending on the bacterial species used and comprised immunomodulatory functions, such as secretion of IL-1α/ß, Il-6, antimicrobial peptides and increased gene transcription of IL-10 and TLR2. The colonisation also influenced the secretion of growth factors such as VFGFA and FGF2. Notably, many of these changes have already previously been associated with the presence of skin commensals. Concomitantly, the model gained first insights on the microbiome's influence on skin xenobiotic metabolism (i.e., CYP1A1, CYP1B1 and CYP2D6) and olfactory receptor expression. The system provides urgently needed experimental access for assessing the toxicological impact of microbiome-associated xenobiotic metabolism in situ.

Mineral oil in food, cosmetic products, and in products regulated by other legislations.

For a few years, mineral oils and their potential adverse health effects have been a constant issue of concern in many regulatory areas such as food, cosmetics, other consumer products, and industrial chemicals. Analytically, two fractions can be distinguished: mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH). This paper aims at assessing the bioaccumulative potential and associated histopathological effects of MOSH as well as the carcinogenic potential of MOAH for consumer-relevant mineral oils. It also covers the absorption, distribution, metabolism, and excretion of MOSH and MOAH upon oral and dermal exposures. The use and occurrence of consumer-relevant, highly refined mineral oils in food, cosmetics and medicinal products are summarized, and estimates for the exposure of consumers are provided. Also addressed are the challenges in characterizing the substance identity of mineral oil products under REACH. Evidence from more recent autopsy and biopsy studies, along with information on decreasing food contamination levels, indicates a low risk for adverse hepatic lesions that may arise from the retention of MOSH in the liver. With respect to MOAH, at present there is no indication of any carcinogenic effects in animals dermally or orally exposed to highly refined mineral oils and waxes. Such products are used not only in cosmetics but also in medicinal products and as additives in food contact materials. The safety of these mineral oil-containing products is thus indirectly documented by their prevalent and long-term use, with a simultaneous lack of clinical and epidemiological evidence for adverse health effects.

Aggregated aluminium exposure: risk assessment for the general population.

Aluminium is one of the most abundant elements in earth's crust and its manifold uses result in an exposure of the population from many sources. Developmental toxicity, effects on the urinary tract and neurotoxicity are known effects of aluminium and its compounds. Here, we assessed the health risks resulting from total consumer exposure towards aluminium and various aluminium compounds, including contributions from foodstuffs, food additives, food contact materials (FCM), and cosmetic products. For the estimation of aluminium contents in foodstuff, data from the German "Pilot-Total-Diet-Study" were used, which was conducted as part of the European TDS-Exposure project. These were combined with consumption data from the German National Consumption Survey II to yield aluminium exposure via food for adults. It was found that the average weekly aluminium exposure resulting from food intake amounts to approx. 50% of the tolerable weekly intake (TWI) of 1 mg/kg body weight (bw)/week, derived by the European Food Safety Authority (EFSA). For children, data from the French "Infant Total Diet Study" and the "Second French Total Diet Study" were used to estimate aluminium exposure via food. As a result, the TWI can be exhausted or slightly exceeded-particularly for infants who are not exclusively breastfed and young children relying on specially adapted diets (e.g. soy-based, lactose free, hypoallergenic). When taking into account the overall aluminium exposure from foods, cosmetic products (cosmetics), pharmaceuticals and FCM from uncoated aluminium, a significant exceedance of the EFSA-derived TWI and even the PTWI of 2 mg/kg bw/week, derived by the Joint FAO/WHO Expert Committee on Food Additives, may occur. Specifically, high exposure levels were found for adolescents aged 11-14 years. Although exposure data were collected with special regard to the German population, it is also representative for European and comparable to international consumers. From a toxicological point of view, regular exceedance of the lifetime tolerable aluminium intake (TWI/PTWI) is undesirable, since this results in an increased risk for health impairments. Consequently, recommendations on how to reduce overall aluminium exposure are given.

Skin toxicology and 3Rs-Current challenges for public health protection.

Driven by the fast paced development of complex test systems in vitro, mass spectrometry and omics, we finally have the tools to unravel the molecular events that underlie toxicological adversity. Yet, timely regulatory adaptation of these new tools continues to pose major challenges even for organs readily accessible such as skin. The reasons for this encompass a need for conservatism as well as the need of tests to serve an existing regulatory framework rather than to produce scientific knowledge. It is important to be aware of this in order to align regulatory skin toxicity with the 3R principles more readily. While most chemical safety testing is still based on animal data, regulatory frameworks have seen a strong push towards non-animal approaches. The endpoints corrosion, irritation, sensitisation, absorption and phototoxicity, for example, can now be covered in vitro with the corresponding test guidelines (TGs) being made available by the OECD. However, in vitro approaches tend to be more reductionist. Hence, a combination of several tests is usually preferable to achieve satisfying predictivity. Moreover, the test systems and their combined use need to be standardised and are therefore subject not only to validation but also to the ongoing development of so-called integrated approaches to testing and assessment (IATAs). Concomitantly, skin models are being refined to deliver the complexity required for increased applicability and predictivity. Given the importance of regulatory applicability for 3R-derived approaches to have a long-lasting impact, this review examines the state of regulatory implementation and perspectives, respectively.

A medical-toxicological view of tattooing.

Long perceived as a form of exotic self-expression in some social fringe groups, tattoos have left their maverick image behind and become mainstream, particularly for young people. Historically, tattoo-related health and safety regulations have focused on rules of hygiene and prevention of infections. Meanwhile, the increasing popularity of tattooing has led to the development of many new colours, allowing tattoos to be more spectacular than ever before. However, little is known about the toxicological risks of the ingredients used. For risk assessment, safe intradermal application of these pigments needs data for toxicity and biokinetics and increased knowledge about the removal of tattoos. Other concerns are the potential for phototoxicity, substance migration, and the possible metabolic conversion of tattoo ink ingredients into toxic substances. Similar considerations apply to cleavage products that are formed during laser-assisted tattoo removal. In this Review, we summarise the issues of concern, putting them into context, and provide perspectives for the assessment of the acute and chronic health effects associated with tattooing.

A Novel Dual-Color Luciferase Reporter Assay for Simultaneous Detection of Estrogen and Aryl Hydrocarbon Receptor Activation.

Consumers are exposed to a plethora of anthropogenic and natural substances that can act as agonists or antagonists for various transcription factors. Depending on the exposure and potency, such interactions can potentially lead to adverse health effects, particularly for substances with multiple molecular targets. The early detection of such interactions is thus of high toxicological interest. Here, we report on the development of a new cellular dual-color reporter assay that allows for time-resolved and quantitative recording of estrogen receptor (ER) and aryl hydrocarbon receptor (AHR) activation in living cells. Both receptors are known for their ligand promiscuity. Moreover, both receptor signaling pathways are interconnected by direct protein-protein interactions as well as by shared protein factors and the competition for ligands. The assay is based on two rare beetle luciferases that emit light in the red (SLR) and green (ELuc) spectrum and that have been stably inserted into human T-47D mammary carcinoma cells. The corresponding cell line is termed "XEER" and has been successfully subjected to proof-of-principle studies using prototypical ER and AHR ligands as well as various phytochemicals, xenobiotics, and extracts from various plastic products.

Combination of Metabolomics with Cellular Assays Reveals New Biomarkers and Mechanistic Insights on Xenoestrogenic Exposures in MCF-7 Cells.

The disruptive potential of xenoestrogens like bisphenol A (BPA) lies in their 17ß-estradiol (E2)-like binding to estrogen receptors (ERs) followed by concomitant modulation of ER target gene expression. Unsurprisingly, most endocrine testing systems focus on the quantification of canonical transcripts or ER-sensitive reporters. However, only little information is available about the corresponding metabolomic changes in vitro. This knowledge gap becomes particularly relevant in the context of potential mixture effects, for example, as a consequence of coexposure to potentially estrogenically active pollutants (e.g., Cd2+). Such effects are often difficult to dissect with molecular tools, especially with regard to potential physiological relevance. Metabolomic biomarkers are well-suited to address this latter aspect as they provide a comprehensive readout of whole-cell physiology. Applying a targeted metabolomics approach (FIA-MS/MS), this study looked for biomarkers indicative of xenoestrogenic exposure in MCF-7 cells. Cells were treated with E2 and BPA in the presence or absence of Cd2+. Statistical analysis revealed a total of 11 amino acids and phospholipids to be related to the compound's estrogenic potency. Co-exposure to Cd2+ modulated the estrogenic profile. However, the corresponding changes were found to be moderate with cellular assays such as the E-screen failing to record any Cd2+-specific estrogenic effects. Overall, metabolomics analysis identified proline as the most prominent estrogenic biomarker. Its increase could clearly be related to estrogenic exposure and concomitant ERα-mediated induction of proliferation. Involvement of the latter was confirmed by siRNA-mediated knockdown studies as well as by receptor inhibition. Further, the underlying signaling was also found to involve the oncoprotein MYC. Taken together, this study provides insights into the underlying mechanisms of xenoestrogenic effects and exemplify the strength of the complementary use of metabolomics and cellular and molecular assays.

Toxification of polycyclic aromatic hydrocarbons by commensal bacteria from human skin.

The ubiquitous occurrence of polycyclic aromatic hydrocarbons (PAHs) leads to constant human exposure at low levels. Toxicologically relevant are especially the high-molecular weight substances due to their (pro-)carcinogenic potential. Following ingestion or uptake, the eukaryotic phase I metabolism often activates these substances to become potent DNA binders, and unsurprisingly metabolism and DNA-adduct formation of model substances such as benzo[a]pyrene (B[a]P) are well studied. However, apart from being subjected to eukaryotic transformations PAHs are also carbon and energy sources for the myriads of commensal microbes inhabiting man's every surface. Yet, we know little about the microbiome's PAH-metabolism capacity and its potentially adverse impact on the human host. This study now shows that readily isolable skin commensals transform B[a]P into a range of highly cyto- and genotoxic metabolites that are excreted in toxicologically relevant concentrations during growth. The respective bacterial supernatants contain a mixture of established eukaryotic as well as hitherto unknown prokaryotic metabolites, the combination of which leads to an increased toxicity. Altogether we show that PAH metabolism of the microbiome has to be considered a potential hazard.

The challenge of the application of 'omics technologies in chemicals risk assessment: Background and outlook.

This survey by the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) highlights that 'omics technologies are generally not yet applied to meet standard information requirements during regulatory hazard assessment. While they are used within weight-of-evidence approaches to investigate substances' modes-of-action, consistent approaches for the generation, processing and interpretation of 'omics data are not applied. To date, no 'omics technology has been standardised or validated. Best practices for performing 'omics studies for regulatory purposes (e.g., microarrays for transcriptome profiling) remain to be established. Therefore, three frameworks for (i) establishing a Good-Laboratory Practice-like context for collecting, storing and curating 'omics data; (ii) 'omics data processing; and (iii) quantitative WoE approaches to interpret 'omics data have been developed, that are presented in this journal supplement. Application of the frameworks will enable between-study comparison of results, which will facilitate the regulatory applicability of 'omics data. The frameworks do not constitute prescriptive protocols precluding any other data analysis method, but provide a baseline for analysis that can be applied to all data allowing ready cross-comparison. Data analysis that does not follow the frameworks can be justified and the resulting data can be compared with the Framework-based common analysis output.