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The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA’s 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU ‘on the ground’ in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

International audience; SCCS (Scientific Committee on Consumer Safety), scientific opinion on Butylated hydroxytoluene (BHT), preliminary version of September 27, 2021, final version of December 2, 2021, SCCS/1636/21.

Risk assessments of various types of chemical compounds are carried out in the European Union (EU) foremost to comply with legislation and to support regulatory decision-making with respect to their safety. Historically, risk assessment has relied heavily on animal experiments. However, the EU is committed to reduce animal experimentation and has implemented several legislative changes, which have triggered a paradigm shift towards human-relevant animal-free testing in the field of toxicology, in particular for risk assessment. For some specific endpoints, such as skin corrosion and irritation, validated alternatives are available whilst for other endpoints, including repeated dose systemic toxicity, the use of animal data is still central to meet the information requirements stipulated in the different legislations. The present review aims to provide an overview of established and more recently introduced methods for hazard assessment and risk characterisation for human health, in particular in the context of the EU Cosmetics Regulation (EC No 1223/2009) as well as the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) Regulation (EC 1907/2006).

Public awareness and discussion about animal experiments and replacement methods has greatly increased in recent years. The term ‘the Three Rs’, which stands for the Replacement, Reduction and Refinement of animal experiments, is inseparably linked in this context. A common goal within the Three Rs scientific community is to develop predictive non-animal models and to better integrate all available data from in vitro, in silico and omics technologies into regulatory decision-making processes regarding, for example, the toxicity of chemicals, drugs or food ingredients. In addition, it is a general concern to implement (human) non-animal methods in basic research. Toward these efforts, there has been an ever-increasing number of Three Rs centres and platforms established over recent years — not only to develop novel methods, but also to disseminate knowledge and help to implement the Three Rs principles in policies and education. The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes gave a strong impetus to the creation of Three Rs initiatives, in the form of centres and platforms. As the first of a series of papers, this article gives an overview of the European Three Rs centres and platforms, and their historical development. The subsequent articles, to be published over the course of ATLA’s 50th Anniversary year, will summarise the current focus and tasks as well as the future and the plans of the Three Rs centres and platforms. The Three Rs centres and platforms are very important points of contact and play an immense role in their respective countries as ‘on the ground’ facilitators of Directive 2010/63/EU. They are also invaluable for the widespread dissemination of information and for promoting implementation of the Three Rs in general.

Non-alcoholic steatohepatitis (NASH) is a highly prevalent, chronic liver disease characterized by hepatic lipid accumulation, inflammation, and concomitant fibrosis. Up to date, no anti-NASH drugs have been approved. In this study, we reproduced key NASH characteristics in vitro by exposing primary human hepatocytes (PHH), human skin stem cell-derived hepatic cells (hSKP-HPC), HepaRG and HepG2 cell lines, as well as LX-2 cells to multiple factors that play a role in the onset of NASH. The obtained in vitro disease models showed intracellular lipid accumulation, secretion of inflammatory chemokines, induced ATP content, apoptosis, and increased pro-fibrotic gene expression. These cell systems were then used to evaluate the anti-NASH properties of eight peroxisome proliferator-activated receptor (PPAR) agonists (bezafibrate, elafibranor, fenofibrate, lanifibranor, pemafibrate, pioglitazone, rosiglitazone, and saroglitazar). PPAR agonists differently attenuated lipid accumulation, inflammatory chemokine secretion, and pro-fibrotic gene expression.Based on the obtained readouts, a scoring system was developed to grade the anti-NASH potencies. The in vitro scoring system, based on a battery of the most performant models, namely PHH, hSKP-HPC, and LX-2 cultures, showed that elafibranor, followed by saroglitazar and pioglitazone, induced the strongest anti-NASH effects. These data corroborate available clinical data and show the relevance of these in vitro models for the preclinical investigation of anti-NASH compounds.

Introduction Since its introduction, the e-cigarette has become a commonly used consumer product. In this study, we investigate whether regulatory changes had an impact on the quality of refill liquids (e-liquids) available on the Belgian market through analysis of their chemical composition. Hence, the nicotine concentration accuracy was investigated in samples before, during and after the implementation of the revised Tobacco Product Directive (TPD) as an indicator of good manufacturing practices. This is, however, not enough to assure the quality. Therefore, extra criteria were also assessed based on TPD requirements. Methods By using in-house validated methods, a total of 246 e-liquids purchased prior (2013–2015), during (2016) and after (2017–2018) the implementation of the TPD revisions, were analyzed for the presence of nicotine, nicotine-related impurities, volatile organic compounds (VOCs), caffeine and taurine, and the flavors diacetyl and acetylpropionyl. Results Although not all manufacturers managed to produce and label their products accurately, nicotine labeling discrepancies have decreased over time. Moreover, also the number of e-liquids, containing high-risk VOCs (10% in 2016 vs. none of the samples in 2017–2018), caffeine (16% in 2017 vs. 5% in 2018), and diacetyl and acetylpropionyl (50% in 2017 vs. 27% in 2018 of sweet-flavored samples) diminished over time. Conclusion Our results demonstrate that the overall quality of the e-liquids has improved after the implementation of the revised TPD. However, the results also show that periodic quality control might be required to ensure further compliance to the TPD. Implications This study clearly demonstrates that the implementation of the revised TPD has improved the quality of the e-liquids on the Belgian market. However, there are still e-liquids that are not in agreement with the TPD due to nicotine concentration label discrepancies, presence of e-liquid impurities and controversial flavors diacetyl and acetylpropionyl or the additive caffeine.

Although most same-stage non-alcoholic fatty liver disease (NAFLD) patients exhibit similar histologic sequelae, the underlying mechanisms appear to be highly heterogeneous. Therefore, it was recently proposed to redefine NAFLD to metabolic dysfunction-associated fatty liver disease (MAFLD) in which other known causes of liver disease such as alcohol consumption or viral hepatitis do not need to be excluded. Revised nomenclature envisions speeding up and facilitating anti-MAFLD drug development by means of patient stratification whereby each subgroup would benefit from distinct pharmacological interventions. As human-based in vitro research fulfils an irrefutable step in drug development, action should be taken as well in this stadium of the translational path. Indeed, most established in vitro NAFLD models rely on short-term exposure to fatty acids and use lipid accumulation as a phenotypic benchmark. This general approach to a seemingly ambiguous disease such as NAFLD therefore no longer seems applicable. Human-based in vitro models that accurately reflect distinct disease subgroups of MAFLD should thus be adopted in early preclinical disease modeling and drug testing. In this review article, we outline considerations for setting up translational in vitro experiments in the MAFLD era and allude to potential strategies to implement MAFLD heterogeneity into an in vitro setting so as to better align early drug development with future clinical trial designs.

Transcriptomics-based biomarkers are promising new approach methodologies (NAMs) to identify molecular events underlying the genotoxic mode of action of chemicals. Previously, we developed the GENOMARK biomarker, consisting of 84 genes selected based on whole genomics DNA microarray profiles of 24 (non-)genotoxic reference chemicals covering different modes of action in metabolically competent human HepaRG™ cells. In the present study, new prediction models for genotoxicity were developed based on an extended reference dataset of 38 chemicals including existing as well as newly generated gene expression data. Both unsupervised and supervised machine learning algorithms were used, but as unsupervised machine learning did not clearly distinguish both groups, the performance of two supervised machine learning algorithms, i.e., support vector machine (SVM) and random forest (RF), was evaluated. More specifically, the predictive accuracy was compared, the sensitivity to outliers for one or more biomarker genes was assessed, and the prediction performance for 10 misleading positive chemicals exposed at their IC10 concentration was determined. In addition, the applicability of both prediction models on a publicly available gene expression dataset, generated with RNA-sequencing, was investigated. Overall, the RF and SVM models were complementary in their classification of chemicals for genotoxicity. To facilitate data analysis, an online application was developed, combining the outcomes of both prediction models. Furthermore, this research demonstrates that the combination of gene expression data with supervised machine learning algorithms can contribute to the ongoing paradigm shift towards a more human-relevant in vitro genotoxicity testing strategy without the use of experimental animals.

Implementation of hydrogel precursors in two-photon polymerization (2PP) technology provides promising opportunities in the tissue engineering field thanks to their soft characteristics and similarity to extracellular matrix. Most of the hydrogels, however, are prone to post-fabrication deformations, leading to a mismatch between the computer-aided design and the printed structure. In the present work, we have developed novel synthetic hydrogel precursors to overcome the limitations associated with 2PP processing of conventional hydrogel precursors such as post-processing deformations and a narrow processing window. The precursors are based on a poly(ethylene glycol) backbone containing urethane linkers and are, on average, functionalized with six acrylate terminal groups (three on each terminal group). As a benchmark material, we exploited a precursor with an identical backbone and urethane linkers, albeit functionalized with two acrylate groups, that were reported as state-of-the-art. An in-depth characterization of the hexafunctional precursors revealed a reduced swelling ratio (36 MPa Young's modulus) compared to their difunctional analogs. The superior physical properties of the newly developed hydrogels lead to 2PP-based fabrication of stable microstructures with excellent shape fidelity at laser scanning speeds up to at least 90 mm s-1, in contrast with the distorted structures of conventional difunctional precursors. The hydrogel films and microscaffolds revealed a good cell interactivity after functionalization of their surface with a gelatin methacrylamide-based coating. The proposed synthesis strategy provides a one-pot and scalable synthesis of hydrogel building blocks that can overcome the current limitations associated with 2PP fabrication of hydrogel microstructures

The Cosmetic Regulation (EC) No 1223/2009 specifically covers the risk of nanomaterials used in cosmetic products. If there are concerns regarding the safety of a nanomaterial, the European Commission refers it to the SCCS for a scientific opinion. The Commission mandated the SCCS to identify the scientific basis for safety concerns that could be used as a basis for identifying and prioritising nanomaterials for safety assessment, and to revisit previous inconclusive SCCS opinions on nanomaterials to identify any concerns for potential risks to the consumer health. The SCCS Scientific Advice identified the key general aspects of nanomaterials that should raise a safety concern for a safety assessor/manager, so that the nanomaterial(s) in question could be subjected to safety assessment to establish safety to the consumer. The Advice also developed a list of the nanomaterials notified to the Commission for use in cosmetics in an order of priority for safety assessment, and revisited three previous inconclusive opinions on nanomaterials to highlight concerns over consumer safety that merited further safety assessment.

Allergic contact dermatitis (ACD) often associated with the topical use of perfumed products, remains one of the most common chronic skin disorders in Western countries. Since labelling of scented menstrual hygiene products (MHPs) is not mandatory, women might be unknowingly exposed to allergens. Given that vaginal mucosae lack the vital barrier function of the skin, skin allergens can easily penetrate and become systemically available and hence women may experience adverse effects in the anogenital region. The aim of this study was therefore to investigate whether women using scented MHPs are at risk of sensitization and hence developing ACD. Hereto, a Quantitative Risk Assessment (QRA) is performed on four well-known skin sensitizing chemicals (α-isomethyl ionone, benzyl salicylate, hexyl cinnamaldehyde and heliotropine) that were previously found leaching from five different scented MHPs including tampons and sanitary pads. The amounts of heliotropine, leached by one of the investigated tampons, exceeded acceptable exposure levels determined with the QRA and could induce sensitization. In addition, although no sensitization is expected for the other three compounds, an allergenic reaction might be provoked in women who are already sensitized. Labelling of allergens on scented MHPs would therefore help consumers to prevent adverse effects linked to ACD.

Correction to: Arch Toxicol (2014) 88:2261–2287 https://doi.org/10.1007/s00204-014-1400-x

In cosmetic products, the ingredient propylparaben (CAS No 94-13-3, EC No 202-307-7) with the chemical names Propyl 4-hydroxybenzoate and 4-Hydroxybenzoic acid propyl ester is currently regulated as a preservative in a concentration up to 0.14% (as acid) (Annex V/12a). In addition, a safe concentration was established for mixtures of parabens, where the sum of the individual concentrations should not exceed 0.8% (as acid). However, in such mixtures the sum of the individual concentrations of butyl- and propylparaben and their salts should not exceed 0.14%. Propylparaben was subject to different safety evaluations in 2005 (SCCP/0874/05), 2006 (SCCP/1017/06), 2008 (SCCP/1183/08), 2010 (SCCS/1348/10), 2011 (SCCS/1446/11), and in 2013 (SCCS/1514/13). On the basis of the safety assessment of propylparaben, and considering the concerns related to potential endocrine disrupting properties, the SCCS has concluded that propylparaben is safe when used as a preservative in cosmetic products up to a maximum concentration of 0.14%. The available data on propylparaben provide some indications for potential endocrine effects. However, the current level of evidence is not sufficient to regard it as an endocrine disrupting substance, or to derive a toxicological point of departure based on endocrine disrupting properties for use in human health risk assessment. The SCCS mandate does not address environmental aspects. Therefore, this assessment did not cover the safety of propylparaben for the environment. Link to the Opinion (SCCS/1623/20): https://ec.europa.eu/health/sites/default/files/scientific_committees/consumer_safety/docs/sccs_o_243.pdf

Adult human subcutaneous adipose tissue (AT) harbors a rich population of mesenchymal stromal cells (MSCs) that are of interest for tissue repair. For this purpose, it is of utmost importance to determine the response of AT-MSCs to proliferative and inflammatory signals within the damaged tissue. We have characterized the transcriptional profile of cytokines, regulatory mediators and Toll-like receptors (TLR) relevant to the response of MSCs. AT-MSCs constitutively present a distinct profile for each gene and differentially responded to inflammation and cell-passaging. Inflammation leads to an upregulation of IL-6, IL-8, IL-1β, TNFα and CCL5 cytokine expression. Inflammation and cell-passaging increased the expression of HGF, IDO1, PTGS1, PTGS2 and TGFβ. The expression of the TLR pattern was differentially modulated with TLR 1, 2, 3, 4, 9 and 10 being increased, whereas TLR 5 and 6 downregulated. Functional enrichment analysis demonstrated a complex interplay between cytokines, TLR and regulatory mediators central for tissue repair. This profiling highlights that following a combination of inflammatory and proliferative signals, the sensitivity and responsive capacity of AT-MSCs may be significantly modified. Understanding these transcriptional changes may help the development of novel therapeutic approaches., SCOPUS: ar.j, info:eu-repo/semantics/published

The electronic cigarette (e-cigarette) has emerged as a popular alternative to the traditional hazardous tobacco cigarette. The substantial increase in e-cigarette use also urgently calls for controlling the quality of e-cigarette refill liquid products (e-liquids). Currently, the most important quality indicator of e-liquid products is the quantification of nicotine and its related impurities. Although different methods have been published to measure nicotine and impurity levels, the majority of them use a targeted LC-MS/MS approach. There is, however, a need for more robust quantification methods that are easy to implement in most control (industrial and governmental) laboratories. Therefore, in this study, a simple dilute-and-shoot UHPLC-DAD method has been developed and validated for the simultaneous quantification of nicotine and its alkaloid impurities in electronic cigarette refills. An optimal separation of the alkaloids was achieved in a runtime of 11 min. The method was successfully validated using the “total error” approach in accordance with the validation requirements of ISO-17025. During this validation, interference between the target components and a number of popular flavouring compounds such as vanillin, maltol, ethylacetate, etc. could be excluded. In addition, small changes to the column temperature, pH and molar concentration of the mobile phase buffer were deliberately introduced in order to assess the robustness of the method. Only a slightly different outcome between the newly developed UV-detection method and the targeted MS approach was found, due to the sensitivity of the different detection techniques. However, in the context of quality control of nicotine related impurities, for which the European Pharmacopoeia limits are currently applied, the sensitivity of the UHPLC-DAD method was found to be within the acceptable range. Despite the somewhat lower selectivity of the newly developed UV-detection technique versus a targeted LC-MS/MS approach, it may be concluded that this method is a suitable alternative for quality control purposes.

Stem cells are characterized by their self-renewal capacity and their ability to differentiate into multiple cell types of the human body. Using directed differentiation strategies, stem cells can now be converted into hepatocyte-like cells (HLCs) and therefore, represent a unique cell source for toxicological applications in vitro. However, the acquired hepatic functionality of stem cell-derived HLCs is still significantly inferior to primary human hepatocytes. One of the main reasons for this is that most in vitro models use traditional two-dimensional (2D) setups where the flat substrata cannot properly mimic the physiology of the human liver. Therefore, 2D-setups are progressively being replaced by more advanced culture systems, which attempt to replicate the natural liver microenvironment, in which stem cells can better differentiate towards HLCs. This review highlights the most recent cell culture systems, including scaffold-free and scaffold-based three-dimensional (3D) technologies and microfluidics that can be employed for culture and hepatic differentiation of stem cells intended for hepatotoxicity testing. These methodologies have shown to improve in vitro liver cell functionality according to the in vivo liver physiology and allow to establish stem cell-based hepatic in vitro platforms for the accurate evaluation of xenobiotics.

Next generation risk assessment (NGRA) is an exposure-led, hypothesis-driven approach that has the potential to support animal-free safety decision-making. However, significant effort is needed to develop and test the in vitro and in silico (computational) approaches that underpin NGRA to enable confident application in a regulatory context. A workshop was held in Montreal in 2019 to discuss where effort needs to be focussed and to agree on the steps needed to ensure safety decisions made on cosmetic ingredients are robust and protective. Workshop participants explored whether NGRA for cosmetic ingredients can be protective of human health, and reviewed examples of NGRA for cosmetic ingredients. From the limited examples available, it is clear that NGRA is still in its infancy, and further case studies are needed to determine whether safety decisions are sufficiently protective and not overly conservative. Seven areas were identified to help progress application of NGRA, including further investments in case studies that elaborate on scenarios frequently encountered by industry and regulators, including those where a 'high risk' conclusion would be expected. These will provide confidence that the tools and approaches can reliably discern differing levels of risk. Furthermore, frameworks to guide performance and reporting should be developed.

Menstrual hygiene products (MHPs) like tampons, sanitary towels and panty liners are widely used by women and come in close contact with the intimate parts of the human body, which consist of mucosae that lack the important barrier function of normal skin. Hence, substances leaching from MHP can easily penetrate and become systemically available. This study aims to develop a new in chemico methodology that allows to identify and measure realistic consumer exposure levels of several skin sensitizers and irritants leaching from MHPs under simulated use conditions. To assess the leaching of chemicals from MHPs, a menstrual fluid simulant (MFS) simulating pH, osmolarity and protein binding was first established. Subsequently, an analytical methodology was developed for nine well-known skin sensitizers and skin irritants. In short, the MFS samples underwent salting-out assisted liquid-liquid extraction before ultra-high performance liquid chromatography coupled with a triple-quadrupole mass spectrometry analysis. Validation was performed according to the total error approach with acceptability limits of ±15% regarding the total analytical error (including systematic and random bias). Fifteen commercially available MHPs were assessed. Six products were found to leach at least one of the following five sensitizing and irritating compounds: α-isomethyl ionone, benzyl salicylate, hexyl cinnamaldehyde, linalool and piperonal. Piperonal was the most abundant compound leaching from the MHPs, with leaching concentration levels measured to 28.22 μg/g. In addition, the leaching level of benzyl salicylate was found to be 11.03 μg/g. The latter fragrance concentration is above 10 μg/g and would trigger mandatory labelling if the Cosmetic Regulation would apply for MHPs. However, none of the identified and quantified skin sensitizers were mentioned on the packaging. In conclusion, this novel methodology makes it possible to estimate realistic human exposure levels to skin sensitizers and irritants through the use of MHPs. Availability of these exposure estimates is vital to carry out a quantitative health risk assessment of these substances.

Metabolic-associated fatty liver disease (MAFLD) is a chronic liver disease that affects about a quarter of the world population. MAFLD encompasses different disease stadia ranging from isolated liver steatosis to non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis and hepatocellular carcinoma. Although MAFLD is considered as the hepatic manifestation of the metabolic syndrome, multiple concomitant disease-potentiating factors can accelerate disease progression. Among these risk factors are diet, lifestyle, genetic traits, intake of steatogenic drugs, male gender and particular infections. Although infections often outweigh the development of fatty liver disease, pre-existing MAFLD could be triggered to progress towards more severe disease stadia. These combined disease cases might be underreported because of the high prevalence of both MAFLD and infectious diseases that can promote or exacerbate fatty liver disease development. In this review, we portray the molecular and cellular mechanisms by which the most relevant viral, bacterial and parasitic infections influence the progression of fatty liver disease and steatohepatitis. We focus in particular on how infectious diseases, including coronavirus disease-19, hepatitis C, acquired immunodeficiency syndrome, peptic ulcer and periodontitis, exacerbate MAFLD. We specifically underscore the synergistic effects of these infections with other MAFLD-promoting factors.

A focal point in the safety evaluation of cosmetic ingredients includes oral repeated dose toxicity testing, which is intended to address the most complex human endpoints. Seven years after the full implementation of the animal testing ban for cosmetic ingredients in the EU, there are still no alternative methods available capable of fully replacing oral repeated dose toxicity testing. Until this issue is resolved, the development of new cosmetic ingredients remains seriously hampered. The present paper describes a thorough screening of the oral repeated dose toxicity data included in safety evaluation reports of cosmetic ingredients addressed in the Annexes of the Cosmetics Regulation (EC) No 1223/2009, issued by the Scientific Committee on Consumer Safety between 2009 and 2019. The liver and the haematological system were identified as the potentially most frequently affected organs upon oral administration of cosmetic ingredients to animals. Evaluation of altered biochemical, morphological, and histopathological parameters related to hepatotoxicity indicated that the most recurrent events are liver weight changes, elevated liver enzymes, and alterations in serum cholesterol and bilirubin levels. Combined listing of affected parameters associated with steatosis and cholestasis indicated the possible occurrence of cholestasis, provoked by a limited number of cosmetic ingredients. The most frequently affected parameters related to the haematological system were indicative of anaemia. An in-depth analysis allowed characterisation of both regenerative and non-regenerative anaemia, pointing to direct and indirect haematotoxicity, respectively. The results presented in this study call for prioritisation of research targeted towards the development of new approach methodologies fit for animal-free repeated dose toxicity evaluation of cosmetic ingredients. Electronic supplementary material The online version of this article (10.1007/s00204-020-02868-2) contains supplementary material, which is available to authorized users.

The progression of mesenchymal stem cell-based therapy from concept to cure closely depends on the optimization of conditions that allow a better survival and favor the cells to achieve efficient liver regeneration. We have previously demonstrated that adult-derived human liver stem/progenitor cells (ADHLSC) display significant features that support their clinical development. The current work aims at studying the impact of a sustained pro-inflammatory environment on the principal biological features of ADHLSC in vitro. METHODS: ADHLSC from passages 4&ndash, 7 were exposed to a cocktail of inflammatory cytokines for 24 h and 9 days and subsequently analyzed for their viability, expression, and secretion profiles by using flow cytometry, RT-qPCR, and antibody array assay. The impact of inflammation on the hepatocytic differentiation potential of ADHLSC was also evaluated. RESULTS: ADHLSC treated with a pro-inflammatory cocktail displayed significant decrease of cell yield at both times of treatment while cell mortality was observed at 9 days post-priming. After 24 h, no significant changes in the immuno-phenotype of ADHLSC expression profile could be noticed while after 9 days, the expression profile of relevant markers has changed both in the basal conditions and after inflammation treatment. Inflammation cocktail enhanced the release of IL-6, IL-8, CCL5, monocyte-chemo-attractant protein-2 and 3, CXCL1/GRO, and CXCL5/ENA78. Furthermore, while IP-10 secretion was increased after 24 h priming, granulocyte macrophage colony-stimulating factor enhanced secretion was noticed after 9 days treatment. Finally, priming of ADHLSC did not affect their potential to differentiate into hepatocyte-like cells. CONCLUSION: These results indicate that ADHLSCs are highly sensitive to inflammation and respond to such signals by adjusting their gene and protein expression. Accordingly, monitoring the inflammatory status of patients at the time of cell transplantation, will certainly help in enhancing ADHLSC safety and efficiency.

International audience; Although the need for non-animal alternatives has been well recognised for the human health hazard assessment of chemicals in general, it has become especially pressing for cosmetic ingredients due to the full implementation of testing and marketing bans on animal testing under the European Cosmetics Regulation. This means that for the safety assessment of cosmetics, the necessary safety data for both the ingredients and the finished product can be drawn from validated (or scientifically-valid), so-called "Replacement methods". In view of the challenges for safety assessment without recourse to animal test data, the Methodology Working Group of the Scientific Committee on Consumer Safety organised a workshop in February 2019 to discuss the key issues in regard to the use of animal-free alternative methods for the safety evaluation of cosmetic ingredients. This perspective article summarises the outcomes of this workshop and reflects on the state-of-the-art and possible way forward for the safety assessment of cosmetic ingredients for which no experimental animal data exist. The use and optimisation of "New Approach Methodology" that could be useful tools in the context of the "Next Generation Risk Assessment" and the strategic framework for safety assessment of cosmetics were discussed in depth.

Non-alcoholic steatohepatitis (NASH) is a severe chronic liver disease that affects 3 to 5 percent of the world population. It is characterized by hepatic lipid accumulation and inflammation and can progress towards fibrosis, cirrhosis and hepatocellular carcinoma. Until today, no drug has been approved for the treatment of NASH. This delay relates to the complex pathogenesis of NASH and also to a lack of appropriate predictive preclinical testing systems. Furthermore, the human specificity of the NASH pathology hampers a fortiori clinical translation of animal studies. Therefore, we recently employed human skin-derived precursors (hSKP) differentiated to hepatocyte-like cells (hSKP-HPC) as a human-relevant cell source for modelling NASH in vitro. Using this in vitro NASH model, it was possible to test novel drugs being developed for anti-NASH therapy, such as elafibranor. Since steatosis is an important aspect of NASH and multiple drugs are being developed to decelerate and reduce lipid accumulation in the liver, we optimized a flow cytometric method for quantifying neutral lipids in ‘NASH’-triggered hSKP-HPC. This methodology enables efficient identification of anti-steatotic properties of new medicines. • NASH-triggered hSKP-HPC robustly accumulate lipids intracellularly. • Flow cytometric quantification of neutral lipids in NASH-triggered hSKP-HPC allows for accurate determination of the steatotic response. • This method enables efficient identification of potential anti-steatotic drugs in a human-specific model, Graphical abstract Image, graphical abstract

"SCCS OPINION on Benzophenone-3 (CAS No 131-57-7, EC No 205-031-5)"SCCS/1625/20 - Preliminary OpinionU. Bernauer, L. Bodin, Q. Chaudhry, P.J. Coenraads, M. Dusinska, J. Ezendam, E. Gaffet, C. L. Galli, B. Granum, E. Panteri, V. Rogiers, Ch. Rousselle, M. Stepnik, T. Vanhaecke, S. Wijnhoven, A. Koutsodimou, W. Uter, N. von GoetzThe SCCS adopted this document by written procedure on 15 December 2020 (77 pages)Mise en ligne le 21 Décembre 2020https://ec.europa.eu/health/sites/health/files/scientific_committees/consumer_safety/docs/sccs_o_247.pdfConclusion of the opinion:(1) In light of the data provided and taking under consideration the concerns related to potential endocrine disrupting properties of Benzophenone-3, does the SCCS consider Benzophenone-3 safe when used as a UV-filter in cosmetic products up to a maximum concentration of 6% and up to 0.5% in cosmetic products to protect product formulation?On the basis of safety assessment, and considering the concerns related to potential endocrine disrupting properties of benzophenone-3 (BP-3), the SCCS has concluded that:a. The use of BP-3 as a UV-filter up to a maximum concentration of 6% in sunscreen products, either in the form of body cream, sunscreen propellant spray or pump spray, is not safe for the consumer.b. The use of BP-3 as a UV-filter up to a maximum concentration of 6% in face cream, hand cream, and lipsticks is safe for the consumer.c. The use of BP-3 up to 0.5% in cosmetic products to protect the cosmetic formulation is safe for the consumer.(2) Alternatively, what is according to the SCCS the maximum concentration considered safe for use of Benzophenone-3 as a UV-filter in cosmetic products?In the SCCS’s opinion, the use of BP-3 as a UV filter in the following sunscreen products is safe for the consumer up to a maximum concentration of:a. 2.2% in body creams, in propellant sprays and in pump sprays, provided that there is no additional use of BP-3 at 0.5% in the same formulation for protecting the cosmetic formulation.b. Where BP-3 is also used at 0.5% in the same formulation, the levels of BP-3 used as UV filter should not exceed 1.7% in body creams, in propellant sprays and in pump sprays.(3) Does the SCCS have any further scientific concerns with regard to the use of Benzophenone-3 in cosmetic products?It needs to be noted that the SCCS has regarded the currently available evidence for endocrine disrupting properties of BP-3 as inconclusive, and at best equivocal. This applies to all of the available data derived from in silico modelling, in vitro tests and in vivo studies, either considered individually or taken together. The SCCS considers that, whilst there are indications from some studies to suggest that BP-3 may have endocrine effects, the evidence is not conclusive enough at present to enable deriving a specific endocrine-related toxicological point of departure for use in safety assessment.________________________________________Keywords:SCCS, scientific opinion, Benzophenone-3, UV filter, Regulation 1223/2009, CAS Number 131-57-7, EC No 205-031-5________________________________________Opinion to be cited as:SCCS (Scientific Committee on Consumer Safety), Opinion on Benzophenone-3 (CAS No 131-57-7, EC No 205-031-5), preliminary version of 15 December 2020, SCCS/1625/20.

A large number of computer-based prediction methods to determine the potential of chemicals to induce mutations at the gene level has been developed over the last decades. Conversely, only few such methods are currently available to predict potential structural and numerical chromosome aberrations. Even fewer of these are based on the preferred testing method for this endpoint, i.e. the micronucleus test. For the present work, in vivo micronucleus test results of 718 structurally diverse compounds were collected and applied for the construction of new models by means of the freely available SARpy in silico model building software. Multiple QSAR models were created using parameter variation and manual verification of (non-) alerting structures. To this extent, the original set of 718 compounds was split into a training (80 %) and a test (20 %) set. SARpy was applied on the training set to automatically extract sets of rules by generating and selecting substructures based on their prediction performance whereas the test set was used to evaluate model performance. Five different splits were made randomly, each of which had a similar balance between positive and negative substances compared to the full dataset. All generated models were characterised by an overall better performance than existing free and commercial models for the same endpoint, while demonstrating high coverage.

Non-alcoholic fatty liver disease (NAFLD) affects one-third of the population worldwide, of which a substantial number of patients suffer from non-alcoholic steatohepatitis (NASH). NASH is a severe condition characterized by steatosis and concomitant liver inflammation and fibrosis, for which no drug is yet available. NAFLD is also generally conceived as the hepatic manifestation of the metabolic syndrome. Consequently, well-established drugs that are indicated for the treatment of type 2 diabetes and hyperlipidemia are thought to exert effects that alleviate the pathological features of NASH. One class of these drugs targets peroxisome proliferator-activated receptors (PPARs), which are nuclear receptors that play a regulatory role in lipid metabolism and inflammation. Therefore, PPARs are now also being investigated as potential anti-NASH druggable targets. In this paper, we review the mechanisms of action and physiological functions of PPARs and discuss the position of the different PPAR agonists in the therapeutic landscape of NASH. We particularly focus on the PPAR agonists currently under evaluation in clinical phase II and III trials. Preclinical strategies and how refinement and optimization may improve PPAR-targeted anti-NASH drug testing are also discussed. Finally, potential caveats related to PPAR agonism in anti-NASH therapy are stipulated.

Non-alcoholic steatohepatitis (NASH) is a chronic liver disease characterized by excessive triglyceride accumulation in the liver accompanied by inflammation, cell stress and apoptosis. It is the tipping point to the life-threatening stages of non-alcoholic fatty liver disease (NAFLD). Despite the high prevalence of NASH, up to five percent of the global population, there are currently no approved drugs to treat this disease. Animal models, mostly based on specific diets and genetic modifications, are often employed in anti-NASH drug development. However, due to interspecies differences and artificial pathogenic conditions, they do not represent the human situation accurately and are inadequate for testing the efficacy and safety of potential new drugs. Human-based in vitro models provide a more legitimate representation of the human NASH pathophysiology and can be used to investigate the dysregulation of cellular functions associated with the disease. Also in silico methodologies and pathway-based approaches using human datasets, may contribute to a more accurate representation of NASH, thereby facilitating the quest for new anti-NASH drugs. In this review, we describe the molecular components of NASH and how human-based tools can contribute to unraveling the pathogenesis of this disease and be used in anti-NASH drug development. We also propose a roadmap for the development and application of human-based approaches for future investigation of NASH.

Bosentan is well known to induce cholestatic liver toxicity in humans. The present study was set up to characterize the hepatotoxic effects of this drug at the transcriptomic, proteomic, and metabolomic levels. For this purpose, human hepatoma-derived HepaRG cells were exposed to a number of concentrations of bosentan during different periods of time. Bosentan was found to functionally and transcriptionally suppress the bile salt export pump as well as to alter bile acid levels. Pathway analysis of both transcriptomics and proteomics data identified cholestasis as a major toxicological event. Transcriptomics results further showed several gene changes related to the activation of the nuclear farnesoid X receptor. Induction of oxidative stress and inflammation were also observed. Metabolomics analysis indicated changes in the abundance of specific endogenous metabolites related to mitochondrial impairment. The outcome of this study may assist in the further optimization of adverse outcome pathway constructs that mechanistically describe the processes involved in cholestatic liver injury. ispartof: ARCHIVES OF TOXICOLOGY vol:92 issue:6 pages:1939-1952 ispartof: location:Germany status: published

The use of e-cigarettes as alternative for tobacco cigarettes has become increasingly popular, even though their safety has not yet been scientifically established. One of the frequently raised concerns is the potential toxicity of certain flavours present in the e-liquids, such as diacetyl and acetylpropionyl. It is therefore important to be able to identify and quantify both compounds. Numerous analytical methods have been published for determining e-liquid compositions, but concerns exist with respect to the lack of analytical evaluation. Hence in this study, a new HS/GC-MS-based method was developed for the screening and quantification of diacetyl and acetylpropionyl in e-liquids. This method was fully validated using the 'total error' approach. The LOQ of the analytical method was 5ppm for diacetyl and acetylpropionyl. The obtained accuracy profiles show that the β-expectation tolerance intervals did not exceed the acceptance limits of±10%, meaning that 95% of future measurements will be included in the [-10%, 10%] bias limits. As proof of applicability, the validated method was successfully applied on a small set of e-liquid samples, indicating that this methodology could be used for routine quality control analyses of e-liquids.

Up till now, no harmonized EU regulation exists on chemicals used in coatings for food contact materials (FCM). Therefore, these substances need to comply with the general provisions of EU Regulation 1935/2004 and, if present, with national legislation. Different inventory lists of compounds that might be present in coatings are available, but for hundreds of these substances, the potential human health impact of their use in FCM coatings has not (recently) been evaluated. Since detailed evaluation of all compounds is not feasible, a pragmatic approach was developed to identify substances with a potential concern for human health. First, an inventory was assembled containing all substances potentially used in coatings. Afterwards, the genotoxic potential of the non-evaluated substances was predicted in silico using two structure-activity relationship (SAR) software programs. For substances yielding structural alerts in both models, genotoxicity data were collected from previous European evaluations in a non-FCM context and from the European CHemicals Agency (ECHA) website. In total, 53 substances were identified as genotoxic in both in silico models, of which ten were considered to be of high concern. For most of the substances, additional toxicological information is needed.