Adverse outcome pathway-based analysis of liver steatosis in vitro using human liver cell lines.

Here, we present an in vitro test battery to analyze chemicals for their potential to induce liver triglyceride accumulation, a hallmark of liver steatosis. We describe steps for using HepG2 and HepaRG human hepatoma cells in conjunction with a combination of several in vitro assays covering the different molecular initiating events and key events of the respective adverse outcome pathway. This protocol is suitable for assessing single substance effects as well as mixtures allowing their classification as steatotic or non-steatotic. For complete details on the use and execution of this protocol, please refer to Luckert et al. (2018),1 Lichtenstein et al. (2020),2 and Knebel et al. (2019).3.

Mixture effects of co-formulants and two plant protection products in a liver cell line.

Plant protection products (PPPs) consist of one or more active substances and several co-formulants. Active substances provide the functionality of the PPP and are consequently evaluated according to standard test methods set by legal data requirements before approval, whereas co-formulants' toxicity is not as comprehensively assessed. However, in some cases mixture effects of active substances and co-formulants might result in increased or different forms of toxicity. In a proof-of-concept study we hence built on previously published results of Zahn et al. (2018[38]) on the mixture toxicity of Priori Xtra® and Adexar® to specifically investigate the influence of co-formulants on the toxicity of these commonly used fungicides. Products, their respective active substances in combination as well as some co-formulants were applied to human hepatoma cell line (HepaRG) in several dilutions. Cell viability analysis, mRNA expression, abundance of xenobiotic metabolizing enzymes and intracellular concentrations of active substances determined by LC-MS/MS analyses demonstrated that the toxicity of the PPPs is influenced by the presence of co-formulants in vitro. PPPs were more cytotoxic than the mix of their active substances. Gene expression profiles of cells treated with the PPPs were similar to those treated with their respective mixture combinations with marked differences. Co-formulants can cause gene expression changes on their own. LC-MS/MS analyses revealed higher intracellular concentrations of active substances in cells treated with PPPs compared to those treated with the respective active substances' mix. Proteomic data showed co-formulants can induce ABC transporters and CYP enzymes. Co-formulants can contribute to the observed increased toxicity of PPPs compared to their active substances in combination due to kinetic interactions, necessitating a more comprehensive evaluation approach.

Commensal-Related Changes in the Epidermal Barrier Function Lead to Alterations in the Benzo[a]Pyrene Metabolite Profile and Its Distribution in 3D Skin.

Polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene (B[a]P) are among the most abundant environmental pollutants, resulting in continuous exposure of human skin and its microbiota. However, effects of the latter on B[a]P toxicity, absorption, metabolism, and distribution in humans remain unclear. Here, we demonstrate that the skin microbiota does metabolize B[a]P on and in human skin in situ, using a recently developed commensal skin model. In this model, microbial metabolism leads to high concentrations of known microbial B[a]P metabolites on the surface as well as in the epidermal layers. In contrast to what was observed for uncolonized skin, B[a]P and its metabolites were subject to altered rates of skin penetration and diffusion, resulting in up to 58% reduction of metabolites recovered from basal culture medium. The results indicate the reason for this altered behavior to be a microbially induced strengthening of the epidermal barrier. Concomitantly, colonized models showed decreased formation and penetration of the ultimate carcinogen B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), leading, in consequence, to fewer BPDE-DNA adducts being formed. Befittingly, transcript and expression levels of key proteins for repairing environmentally induced DNA damage such as xeroderma pigmentosum complementation group C (XPC) were also found to be reduced in the commensal models, as was expression of B[a]P-associated cytochrome P450-dependent monooxygenases (CYPs). The results show that the microbiome can have significant effects on the toxicology of external chemical impacts. The respective effects rely on a complex interplay between microbial and host metabolism and microbe-host interactions, all of which cannot be adequately assessed using single-system studies. IMPORTANCE Exposure to xenobiotics has repeatedly been associated with adverse health effects. While the majority of reported cases relate to direct substance effects, there is increasing evidence that microbiome-dependent metabolism of xenobiotic substances likewise has direct adverse effects on the host. This can be due to microbial biotransformation of compounds, interaction between the microbiota and the host's endogenous detoxification enzymes, or altered xenobiotic bioavailability. However, there are hardly any studies addressing the complex interplay of such interactions in situ and less so in human test systems. Using a recently developed microbially competent three-dimensional (3D) skin model, we show here for the first time how commensal influence on skin physiology and gene transcription paradoxically modulates PAH toxicity.

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.

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.

Chemical activation of estrogen and aryl hydrocarbon receptor signaling pathways and their interaction in toxicology and metabolism.

INTRODUCTION: Estrogen receptors (ERs) and the arylhydrocarbon receptor (AHR) are ligand-activated transcription factors that regulate the expression of genes involved in many physiological processes. With both receptors binding a broad range of natural and anthropogenic ligands, they are molecular targets for many substances, raising concerns for possible health effects. Areas covered: This review shall give a brief overview on the physiological functions of both receptors including their underlying molecular mechanisms. It summarizes the interaction of the respective signaling pathways including impacts on metabolism of endogenous estrogens, transcriptional interference, inhibitory crosstalk, and proteasomal degradation. Also addressed are the AHR dependent formation of estrogenic metabolites from polycyclic aromatic hydrocarbons and the possible impact of the ER/AHR crosstalk in the context of drug metabolism. Expert opinion: Despite decade-long research, the physiological role of the AHR and ER as well as the implications of their complex mutual crosstalk remain to be determined as do resulting potential impacts on human health. With more and more endogenous AHR ligands being discovered, future research should hence systematically address the potential impact of such substances on estrogen signaling. The intimate link between these two pathways and the genes regulated therein bears the potential for impacts on drug metabolism and human health.

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.

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.

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.

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.

Estrogenic Activity of Mineral Oil Aromatic Hydrocarbons Used in Printing Inks.

The majority of printing inks are based on mineral oils (MOs) which contain complex mixtures of saturated and aromatic hydrocarbons. Consumer exposure to these oils occurs either through direct skin contacts or, more frequently, as a result of MO migration into the contents of food packaging that was made from recycled newspaper. Despite this ubiquitous and frequent exposure little is known about the potential toxicological effects, particularly with regard to the aromatic MO fractions. From a toxicological point of view the huge amount of alkylated and unsubstituted compounds therein is reason for concern as they can harbor genotoxicants as well as potential endocrine disruptors. The aim of this study was to assess both the genotoxic and estrogenic potential of MOs used in printing inks. Mineral oils with various aromatic hydrocarbon contents were tested using a battery of in vitro assays selected to address various endpoints such as estrogen-dependent cell proliferation, activation of estrogen receptor α or transcriptional induction of estrogenic target genes. In addition, the comet assay has been applied to test for genotoxicity. Out of 15 MOs tested, 10 were found to potentially act as xenoestrogens. For most of the oils the effects were clearly triggered by constituents of the aromatic hydrocarbon fraction. From 5 oils tested in the comet assay, 2 showed slight genotoxicity. Altogether it appears that MOs used in printing inks are potential endocrine disruptors and should thus be assessed carefully to what extent they might contribute to the total estrogenic burden in humans.

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.

G protein-coupled receptor 30 ligand G-1 increases aryl hydrocarbon receptor signalling by inhibition of tubulin assembly and cell cycle arrest in human MCF-7 cells.

Regulatory crosstalk between the aryl hydrocarbon receptor (AHR) and oestrogen receptor α (ERα) is well established. Apart from the nuclear receptors ERα and ERß, oestrogen signalling further involves an unrelated G protein-coupled receptor termed GPR30. In order to investigate potential regulatory crosstalk, this study investigated the influence of G-1 as one of the few GPR30-specific ligands on the AHR regulon in MCF-7 cells. As a well-characterised model system, these human mammary carcinoma cells co-express all three receptors (AHR, ERα and GPR30) and are thus ideally suited to study corresponding regulatory pathway interactions on transcript level. Indeed, treatment with micromolar concentrations of the GPR30-specific agonist G-1 resulted in up-regulation of AHR as well as the transcripts for cytochromes P450 1A1 and 1B1, two well-known targets of the AHR regulon. While this was partly attributable to G-1-mediated inhibition of tubulin assembly and subsequent cell cycle arrest in the G2/M phase, the effects nevertheless required functional AHR. However, G-1-induced up-regulation of CYP 1A1 was not mediated by GPR30, as G15 antagonist treatment as well as a knockdown of GPR30 and AHR failed to inhibit this effect.

Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology.

Models of the outer epithelia of the human body - namely the skin, the intestine and the lung - have found valid applications in both research and industrial settings as attractive alternatives to animal testing. A variety of approaches to model these barriers are currently employed in such fields, ranging from the utilization of ex vivo tissue to reconstructed in vitro models, and further to chip-based technologies, synthetic membrane systems and, of increasing current interest, in silico modeling approaches. An international group of experts in the field of epithelial barriers was convened from academia, industry and regulatory bodies to present both the current state of the art of non-animal models of the skin, intestinal and pulmonary barriers in their various fields of application, and to discuss research-based, industry-driven and regulatory-relevant future directions for both the development of new models and the refinement of existing test methods. Issues of model relevance and preference, validation and standardization, acceptance, and the need for simplicity versus complexity were focal themes of the discussions. The outcomes of workshop presentations and discussions, in relation to both current status and future directions in the utilization and development of epithelial barrier models, are presented by the attending experts in the current report.

Insights on the human microbiome and its xenobiotic metabolism: what is known about its effects on human physiology?

INTRODUCTION: Our microbiome harbours a metabolic capacity far beyond our own. Moreover, its gene pool is highly adaptable and subject to selective pressure, including host exposure to xenobiotics. Yet, the resulting adaptations do not necessarily follow host well-being and can therefore contribute to disease or unfavourable metabolite production. AREAS COVERED: This review provides an overview of our host-microbiome relationship in light of bacterial (xenobiotic) metabolism, community dynamics, entero-endocrine crosstalk, dysbiosis and potential therapeutic targets. In addition, it will highlight the need for a systematic analysis of the microbiome's potential for substance toxification. EXPERT OPINION: The influence of our microbiota reaches from primary metabolites to secondary effects such as substrate competition or the activation of eukaryotic Phase I and Phase II enzymes. Further on it plays a hitherto underestimated role in drug metabolism, toxicity and pathogenesis. These effects are partly caused by entero-endocrine crosstalk and interference with eukaryotic regulatory networks. On first sight, the resulting concept of a metabolically competent microbiome adds enormous complexity to human physiology. Yet, the potential specificity of microbial targets harbours therapeutic promise for diseases such as diabetes, cancer and psychiatric disorders. A better physiological and biochemical understanding of the microbiome is thus of high priority for academia and biomedical research.

Degradation of benzo[a]pyrene by bacterial isolates from human skin.

Polycyclic aromatic hydrocarbons (PAHs) are some of the most widespread xenobiotic pollutants, with the potentially carcinogenic high-molecular-weight representatives being of particular interest. However, while in eukaryotes, the cytochrome P450 (CYP)-mediated activation of benzo[a]pyrene (B[a]P) has become a model for metabolism-mediated carcinogenesis, the oxidative degradation of B[a]P by microorganisms is less well studied. This should be reason for concern as the human organ most exposed to environmental PAHs is the skin, which at the same time is habitat to a most diverse population of microbial commensals. Yet, nothing is known about the skin's microbiome potential to metabolise B[a]P. This study now reports on the isolation of 21 B[a]P-degrading microorganisms from human skin, 10 of which were characterised further. All isolates were able to degrade B[a]P as sole source of carbon and energy, and degradation was found to be complete in at least four isolates. Substrate metabolism involved two transcripts that encode a putative DszA/NtaA-like monooxygenase and a NifH-like reductase, respectively. Analysis of the 16S-rRNA genes showed that the B[a]P-degrading isolates comprise Gram(+) as well as Gram(-) skin commensals, with Micrococci being predominant. Moreover, microbial B[a]P-degradation was detected on all volunteers probed, indicating it to be a universal feature of the skin's microbiome.

Effects of triclocarban on the transcription of estrogen, androgen and aryl hydrocarbon receptor responsive genes in human breast cancer cells.

Triclocarban (TCC) is an antimicrobial agent that is used in detergents, soaps and other personal hygiene products. Similarly to triclosan the widespread use of TCC has raised concerns about its endocrine potential. In luciferase-based reporter assays TCC has been shown to enhance estrogenic and androgenic activities following cellular coexposure with estrogen or dihydrotestosterone, respectively. The present study demonstrates that although coexposure with TCC enhances the estrogenic and androgenic readout of luciferase-based reporter cell lines such as HeLa9908 and MDA-kb2, it fails to act as a xenoandrogen on transcriptional level, nor does it induce cell proliferation in the estrogen sensitive E-screen. In addition TCC did not alter the expression of estrogen responsive genes in human mammary carcinoma MCF-7 cells exposed to 17ß-estradiol, bisphenol A, butylparaben or genistein. However, TCC was shown to interfere with the regulon of the aryl hydrocarbon receptor (AhR) as TCC showed a costimulatory effect on transcription of CYP1A1 and CYP1B1, effectively lowering the transcriptional threshold for both genes in the presence of estrogens. It thus seems, that while the induction of the respective luciferase reporter assays by TCC is an unspecific false positive signal caused by luciferase stabilisation, TCC has the potential to interfere with the regulatory crosstalk of the estrogen receptor (ER) and the AhR regulon.

Risk assessment of nanomaterials in cosmetics: a European union perspective.

In Europe, the data requirements for the hazard and exposure characterisation of chemicals are defined according to the REACH regulation and its guidance on information requirements and chemical safety assessment (Regulation (EC) No 1907/2006 of the European Parliament and of the Council of 18 December 2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), and its guidance documents; available at: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2006:396:0001:0849:EN:PDF ; and at: http://guidance.echa.europa.eu/docs/guidance_document/information_requirements_en.htm ). This is the basis for any related risk assessment. The standard reference for the testing of cosmetic ingredients is the SCCP's 'Notes of Guidance for the Testing of Cosmetic Ingredients and their Safety Evaluation' (The SCCP's Notes of Guidance for the testing of cosmetic ingredients and their safety evaluation (2006); available at: http://ec.europa.eu/health/ph_risk/committees/04_sccp/docs/sccp_o_03j.pdf ), which refers to the OECD guidelines for the testing of chemicals (The OECD Guidelines for the Testing of Chemicals as a collection of the most relevant internationally agreed testing methods used by government, industry and independent laboratories to assess the safety of chemical products; available at: http://www.oecd.org/topic/0,2686,en_2649_34377_1_1_1_1_37407,00.html ). According to the cosmetics directive [76/768/EEC], compounds that are classified as mutagenic, carcinogenic or toxic to reproduction are banned for the use in cosmetic products. Since December 2010, the respective labelling is based on the rules of regulation (EC) No. 1272/2008 (Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006, Official Journal L 353, 31/12/2008, pages 1-1355; available at: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2008:353:0001:1355:en:PDF ) on classification, labelling and packaging of substances and mixtures (CLP). There is no further impact from the CLP regulation on cosmetic products, because regulation (EC) No. 1223/2009 on cosmetic products defines its own labelling rules (Regulation (EC) No 1223/2009 of the European Parliament and of the Council of 30 November 2009 on cosmetic products; available at: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2009:342:0059:0209:en:PDF ). Special notification procedures are mandatory for preservatives, colourants and UV-filters where a safety approval from the European 'Scientific Committee on Consumer Safety' (SCCS) is needed prior to marketing. The risk assessment of nanomaterials in consumer products still poses a significant challenge as highlighted by the example of UV-filters in sunscreens since nanomaterials cannot be classified as a homogenous group of chemicals but still need to be addressed in risk characterisation on a case by case basis.

"Drugs on oxygen": an update and perspective on the role of cytochrome P450 testing in pharmacology.

Low hit rates for lead compounds and high attrition remain a major problem for drug development. The reasons for compound failure range from poor pharmacokinetics to toxic metabolites and adverse drug interactions; all of which are frequently mediated by cytochrome P450-dependent monooxygenases (CYPs). However, despite some 30 years of assay development and refinement, CYP metabolism remains a critical issue during drug development. While current testing strategies succeed in characterizing single substance toxicity, they are challenged by practical issues such as assay standardization or complex scenarios such as multidrug usage. This editorial summarizes where we stand and highlights the major challenges we face with CYPs in drug development today. The article also tries to spell out the future direction of CYP testing. The latter will depend on the extended inclusion of polypharmacy into testing strategies, as well as on our capability to make use of upcoming complex in vitro test systems and their inclusion into tiered testing strategies.

Wind of change challenges toxicological regulators.

BACKGROUND: In biomedical research, the past two decades have seen the advent of in vitro model systems based on stem cells, humanized cell lines, and engineered organotypic tissues, as well as numerous cellular assays based on primarily established tumor-derived cell lines and their genetically modified derivatives. OBJECTIVE: There are high hopes that these systems might replace the need for animal testing in regulatory toxicology. However, despite increasing pressure in recent years to reduce animal testing, regulators are still reluctant to adopt in vitro approaches on a large scale. It thus seems appropriate to consider how we could realistically perform regulatory toxicity testing using in vitro assays only. DISCUSSION AND CONCLUSION: Here, we suggest an in vitro-only approach for regulatory testing that will benefit consumers, industry, and regulators alike.