Paving the way for application of next generation risk assessment to safety decision-making for cosmetic ingredients.

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.

Consumer protection provided by the European medical device and cosmetic legislation for condoms and lubricants.

Personal lubricants and lubricants used in condoms contain a number of ingredients which are also present in cosmetic products. These have to comply to the medical device regulation (745/2017) which should provide the same level of consumer protection, if not more, as foreseen in the legal framework of cosmetics (1223/2009). In the current study we developed an analytical method capable of identifying and quantifying 15 ingredients, commonly found in lubricants and cosmetics. Based upon their most important toxicological endpoint, the substances involved were grouped in three toxicological classes provoking either irritation, contact allergic dermatitis or systemic toxicity. The method was applied on 30 condoms and 54 personal lubricants present on the EU market. Their safety was assessed using the same reasoning as commonly applied for cosmetic ingredients. Higher mucosae susceptibility, the main exposed area for lubricants, was taken into account in this assessment. The results show that the majority of the products studied are safe. Nevertheless, for some products the safety could not be confirmed. The results also highlight the fact that there is no consensus for a number of ingredients, used as well in cosmetics as in medical devices. Alignment between both legislations would improve the safety of these products and further raise the general level of consumer protection.

Workshop on acceleration of the validation and regulatory acceptance of alternative methods and implementation of testing strategies.

This report describes the proceedings of the BfR-RIVM workshop on validation of alternative methods which was held 23 and 24 March 2017 in Berlin, Germany. Stakeholders from governmental agencies, regulatory authorities, universities, industry and the OECD were invited to discuss current problems concerning the regulatory acceptance and implementation of alternative test methods and testing strategies, with the aim to develop feasible solutions. Classical validation of alternative methods usually involves one to one comparison with the gold standard animal study. This approach suffers from the reductionist nature of an alternative test as compared to the animal study as well as from the animal study being considered as the gold standard. Modern approaches combine individual alternatives into testing strategies, for which integrated and defined approaches are emerging at OECD. Furthermore, progress in mechanistic toxicology, e.g. through the adverse outcome pathway approach, and in computational systems toxicology allows integration of alternative test battery results into toxicity predictions that are more fine-tuned to the human situation. The road towards transition to a mechanistically-based human-focused hazard and risk assessment of chemicals requires an open mind towards stepping away from the animal study as the gold standard and defining human biologically based regulatory requirements for human hazard and risk assessment.

Coatings in food contact materials: Potential source of genotoxic contaminants?

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.

Overview of skin whitening agents with an insight into the illegal cosmetic market in Europe.

Lightening skin tone is an ancient and well-documented practice, and remains common practice among many cultures. Whitening agents such as corticosteroids, tretinoin and hydroquinone are medically applied to effectively lighten the skin tone of hyperpigmented lesions. However, when these agents are used cosmetically, they are associated with a variety of side-effect. Alternative agents, such as arbutin and its derivatives kojic acid and nicotinamide have been subsequently developed for cosmetic purposes. Unfortunately, some cosmetics contain whitening agents that are banned for use in cosmetic products. This article provides an overview of the mode of action and potential side-effects of cosmetic legal and illegal whitening agents, and the pattern of use of these types of products. Finally, an EU analysis of the health problems due to the presence of illegal products on the market is summarized.

Inter-laboratory study of human in vitro toxicogenomics-based tests as alternative methods for evaluating chemical carcinogenicity: a bioinformatics perspective.

The assessment of the carcinogenic potential of chemicals with alternative, human-based in vitro systems has become a major goal of toxicogenomics. The central read-out of these assays is the transcriptome, and while many studies exist that explored the gene expression responses of such systems, reports on robustness and reproducibility, when testing them independently in different laboratories, are still uncommon. Furthermore, there is limited knowledge about variability induced by the data analysis protocols. We have conducted an inter-laboratory study for testing chemical carcinogenicity evaluating two human in vitro assays: hepatoma-derived cells and hTERT-immortalized renal proximal tubule epithelial cells, representing liver and kidney as major target organs. Cellular systems were initially challenged with thirty compounds, genome-wide gene expression was measured with microarrays, and hazard classifiers were built from this training set. Subsequently, each system was independently established in three different laboratories, and gene expression measurements were conducted using anonymized compounds. Data analysis was performed independently by two separate groups applying different protocols for the assessment of inter-laboratory reproducibility and for the prediction of carcinogenic hazard. As a result, both workflows came to very similar conclusions with respect to (1) identification of experimental outliers, (2) overall assessment of robustness and inter-laboratory reproducibility and (3) re-classification of the unknown compounds to the respective toxicity classes. In summary, the developed bioinformatics workflows deliver accurate measures for inter-laboratory comparison studies, and the study can be used as guidance for validation of future carcinogenicity assays in order to implement testing of human in vitro alternatives to animal testing.

HS-GC-MS method for the analysis of fragrance allergens in complex cosmetic matrices.

Potential allergenic fragrances are part of the Cosmetic Regulation with labelling and concentration restrictions. This means that they have to be declared on the ingredients list, when their concentration exceeds the labelling limit of 10 ppm or 100 ppm for leave-on or rinse-off cosmetics, respectively. Labelling is important regarding consumer safety. In this way, sensitised people towards fragrances might select their products based on the ingredients list to prevent elicitation of an allergic reaction. It is therefore important to quantify potential allergenic ingredients in cosmetic products. An easy to perform liquid extraction was developed, combined with a new headspace GC-MS method. The latter was capable of analysing 24 volatile allergenic fragrances in complex cosmetic formulations, such as hydrophilic (O/W) and lipophilic (W/O) creams, lotions and gels. This method was successfully validated using the total error approach. The trueness deviations for all components were smaller than 8%, and the expectation tolerance limits did not exceed the acceptance limits of ± 20% at the labelling limit. The current methodology was used to analyse 18 cosmetic samples that were already identified as being illegal on the EU market for containing forbidden skin whitening substances. Our results showed that these cosmetic products also contained undeclared fragrances above the limit value for labelling, which imposes an additional health risk for the consumer.

Proposal of an in silico profiler for categorisation of repeat dose toxicity data of hair dyes.

This study outlines the analysis of 94 chemicals with repeat dose toxicity data taken from Scientific Committee on Consumer Safety opinions for commonly used hair dyes in the European Union. Structural similarity was applied to group these chemicals into categories. Subsequent mechanistic analysis suggested that toxicity to mitochondria is potentially a key driver of repeat dose toxicity for chemicals within each of the categories. The mechanistic hypothesis allowed for an in silico profiler consisting of four mechanism-based structural alerts to be proposed. These structural alerts related to a number of important chemical classes such as quinones, anthraquinones, substituted nitrobenzenes and aromatic azos. This in silico profiler is intended for grouping chemicals into mechanism-based categories within the adverse outcome pathway paradigm.

Silicones as nonocclusive topical agents.

BACKGROUND/AIMS: Silicone excipients are commonly used ingredients because of their emollient and skin-conditioning effects, and their ability to form uniform, water-resistant, yet permeable films. Based on comparisons with organic materials and conflicting knowledge from silicones used in scar treatment, the misconception still exists that silicone topical excipients are occlusive substances that may block the passive loss of water through the upper skin layers. Therefore, 3 types of common silicone excipients and 3 water-in-(oil-plus-silicone) or W/(O + Si) creams, containing 10% (w/w) of the respective silicones, were investigated as a function of time and compared to petrolatum. METHODS: Transepidermal water loss (TEWL) and skin hydration measurements were carried out after a single topical application on forearm skin of 26 healthy young female volunteers. RESULTS: Both petrolatum and silicones significantly decreased TEWL 15 min after application, but the measurements for the silicones were not significantly different from the untreated control values. The tested silicones did not moisturize the skin. Petrolatum formed an occlusive layer, creating an increase in skin hydration for more than 4 h. The results measured for the W/(O + Si) creams indicated that they moisturized the skin, without any effect on TEWL. CONCLUSION: A clear difference was shown between the skin occlusive properties of petrolatum and the water vapor permeability of the common silicone excipient materials.

Characterization of suspected illegal skin whitening cosmetics.

An important group of suspected illegal cosmetics consists of skin bleaching products, which are usually applied to the skin of the face, hands and décolleté for local depigmentation of hyper pigmented regions or more importantly, for a generalized reduction of the skin tone. These cosmetic products are suspected to contain illegal active substances that may provoke as well local as systemic toxic effects, being the reason for their banning from the EU market. In that respect, illegal and restricted substances in cosmetics, known to have bleaching properties, are in particular hydroquinone, tretinoin and corticosteroids. From a legislative point of view, all cosmetic products containing a prohibited whitening agent are illegal and must be taken off the EU market. A newly developed screening method using ultra high performance liquid chromatography-time off flight-mass spectrometry allows routine analysis of suspected products. 163 suspected skin whitening cosmetics, collected by Belgian inspectors at high risk sites such as airports and so-called ethnic cosmetic shops, were analyzed and 59% were classified as illegal. The whitening agents mostly detected were clobetasol propionate and hydroquinone, which represent a serious health risk when repeatedly and abundantly applied to the skin.

Practical problems encountered during the cultivation of an open-source reconstructed human epidermis model on a polycarbonate membrane and protein quantification.

During recent years, the importance of in vitro technology in skin research has increased significantly. A variety of skin culture models have been developed and commercialized. In this respect, the availability of reconstructed human epidermis (RHE) equivalents represents a significant improvement compared to the use of monolayer cultures. However, when an in-house RHE model is being developed, researchers might encounter some difficulties during cultivation. The scope of this paper is to report our experiences and practical problems with the development of a three-dimensional RHE model cultured on a polycarbonate membrane. Some important issues including cell density, the use of lysing enzymes, culture media, cell storage and viability, cell confluency and protein extraction are reported and optional solutions are given.

Current status of healthy human skin models: can histone deacetylase inhibitors potentially improve the present replacement models?

Histone deacetylase inhibitors (HDACi), a relatively new group of epigenetic agents, are being investigated as powerful chemotherapeutics because of their antiproliferative and prodifferentiation effects both in vitro and in vivo, in various tumor cell lines. Only little is known with respect to their effects on normal cells. Yet, to understand tissue pathology and evaluate potential effects of new chemical entities in tissue homeostasis, insight into the physiology of healthy tissue is necessary. Therefore, this review addresses the effects of HDACi on healthy human primary skin cell cultures and three-dimensional epidermal models. In general, HDACi exert an effect on both the epidermal morphology and differentiation process of human skin. The latter is manifested through cell cycle arrest, disorganization of the basal layer, thinning of the stratum spinosum and thickening of the stratum corneum, reorganization of the cytoskeleton and increased formation of cornified envelopes. This overview shows that, although only a limited number of reports exist, these molecules might be an interesting tool for the development and study of new human skin models.

Drug-induced liver injury: mechanisms, types and biomarkers.

Drug-induced liver injury is a ubiquitous issue in clinical settings and pharmaceutical industry. Hepatotoxicity elicited by drugs may be intrinsic or idiosyncratic, both which are driven by different molecular mechanisms. Recently, a unifying mechanistic model of drug-induced liver injury has been introduced. According to this model, drug-induced hepatotoxicity relies on 3 consecutive steps, namely an initial cellular insult that leads to the occurrence of mitochondrial permeability transition, which in turn ultimately burgeons into the onset of cell death. Clinically, drug-induced liver injury can be manifested in a number of acute and chronic conditions, including hepatitis, cholestasis, steatosis and fibrosis. These pathologies can be diagnosed and monitored by addressing well-established physical, clinical chemistry and histopathological biomarkers. In the last few years, several novel read-outs of drug-induced liver injury have been proposed, involving genetic, epigenetic, transcriptomic, proteomic and metabolomic parameters. These new concepts and recent developments in the field of drug-induced liver injury are revised in the current paper.

Development and validation of a fast chromatographic method for screening and quantification of legal and illegal skin whitening agents.

During the last years, the EU market is flooded by illegal cosmetics via the Internet and a so-called "black market". Among these, skin-bleaching products represent an important group. They contain, according to the current European cosmetic legislation (Directive 76/768/EEC), a number of illegal active substances including hydroquinone, tretinoin and corticosteroids. These may provoke as well local as systemic toxic effects, being the reason for their banning from the EU market. To control this market there is a need for a fast screening method capable of detecting illegal ingredients in the wide variety of existing bleaching cosmetic formulations. In this paper the development and validation of an ultra high pressure liquid chromatographic (UHPLC) method is described. The proposed method makes use of a Waters Acquity BEH shield RP18 column with a gradient using 25 mM ammonium borate buffer (pH 10) and acetonitrile. This method is not only able to detect the major illegal (hydroquinone, tretinoin and six dermatologic active corticosteroids) and legal whitening agents, the latter having restrictions with respect to concentration and application (kojic acid, arbutin, nicotinamide and salicylic acid), but can also quantify these in a run time of 12 min. The method was successfully validated using the "total error" approach in accordance with the validation requirements of ISO-17025. During the validation a variety of cosmetic matrices including creams, lotions and soaps were taken into consideration.

Primary hepatocyte cultures for pharmaco-toxicological studies: at the busy crossroad of various anti-dedifferentiation strategies.

Continuously increasing understanding of the molecular triggers responsible for the onset of diseases, paralleled by an equally dynamic evolution of chemical synthesis and screening methods, offers an abundance of pharmacological agents with a potential to become new successful drugs. However, before patients can benefit of newly developed pharmaceuticals, stringent safety filters need to be applied to weed out unfavourable drug candidates. Cost effectiveness and the need to identify compound liabilities, without exposing humans to unnecessary risks, has stimulated the shift of the safety studies to the earliest stages of drug discovery and development. In this regard, in vivo relevant organotypic in vitro models have high potential to revolutionize the preclinical safety testing. They can enable automation of the process, to match the requirements of high-throughput screening approaches, while satisfying ethical considerations. Cultures of primary hepatocytes became already an inherent part of the preclinical pharmaco-toxicological testing battery, yet their routine use, particularly for long-term assays, is limited by the progressive deterioration of liver-specific features. The availability of suitable hepatic and other organ-specific in vitro models is, however, of paramount importance in the light of changing European legal regulations in the field of chemical compounds of different origin, which gradually restrict the use of animal studies for safety assessment, as currently witnessed in cosmetic industry. Fortunately, research groups worldwide spare no effort to establish hepatic in vitro systems. In the present review, both classical and innovative methodologies to stabilize the in vivo-like hepatocyte phenotype in culture of primary hepatocytes are presented and discussed.

A liver-derived rat epithelial cell line from biliary origin acquires hepatic functions upon sequential exposure to hepatogenic growth factors and cytokines.

Withdrawal of promising drug candidates is often due to the detection of liver toxicity. In particular the parenchymal liver cells or hepatocytes are targeted since they are the major sites of drug transport and of metabolite formation and thus also the place where not only detoxification, but also activation of new chemical (NCE) and biological (NBE) entities may occur. Therefore, primary hepatocyte- based cultures are currently the preferred in vitro model to screen for liver toxicity. However, within a few days, they undergo dedifferentiation with loss of liver-specific functionality, including xenobiotic biotransformation capacity, making them only suitable for short-term applications. A plausible alternative to primary hepatocyte cultures that can be maintained for longer periods of time could be the use of liver-derived epithelial cell lines and their optimized derivatives. Therefore, in the present study, we evaluated the stability and the hepatic differentiation potential of a neonatal liver-derived rat epithelial cell line from biliary origin (rLEC). Undifferentiated rLEC stably express the hepatic progenitor markers CEBPA, FOXA2, GJA1, ONECUT1, KRT18 and KRT19 for at least 15 consecutive passages after cryopreservation. Upon sequential exposure to hepatogenic growth factors and cytokines, rLEC generate functional hepatic progeny, expressing mature hepatic markers including Alb, Ahr, Car, C/ebpα, Cx32, Foxa2, Hnf1α, Hnf1ß and Onecut1. Furthermore, an active polarization is observed for the hepatic drug transporters Oatp4 and Ntcp. rLEC-derived hepatic cells also acquire the ability to store glycogen, express genes encoding for key hepatic enzymes as shown by Affymetrix microarray data, and display stable CYP1A1/2- and CYP2B1/2-dependent activities for several weeks at levels comparable to those observed in cultured primary rat hepatocytes. The acquisition of such a stable and active biotransformation capacity is key for the applicability of liver-based in vitro models for long-term toxicity testing.

Transfer of IP3 through gap junctions is critical, but not sufficient, for the spread of apoptosis.

Decades of research have indicated that gap junction channels contribute to the propagation of apoptosis between neighboring cells. Inositol 1,4,5-trisphosphate (IP3) has been proposed as the responsible molecule conveying the apoptotic message, although conclusive results are still missing. We investigated the role of IP3 in a model of gap junction-mediated spreading of cytochrome C-induced apoptosis. We used targeted loading of high-molecular-weight agents interfering with the IP3 signaling cascade in the apoptosis trigger zone and cell death communication zone of C6-glioma cells heterologously expressing connexin (Cx)43 or Cx26. Blocking IP3 receptors or stimulating IP3 degradation both diminished the propagation of apoptosis. Apoptosis spread was also reduced in cells expressing mutant Cx26, which forms gap junctions with an impaired IP3 permeability. However, IP3 by itself was not able to induce cell death, but only potentiated cell death propagation when the apoptosis trigger was applied. We conclude that IP3 is a key necessary messenger for communicating apoptotic cell death via gap junctions, but needs to team up with other factors to become a fully pro-apoptotic messenger.

Report from the EPAA workshop: in vitro ADME in safety testing used by EPAA industry sectors.

There are now numerous in vitro and in silico ADME alternatives to in vivo assays but how do different industries incorporate them into their decision tree approaches for risk assessment, bearing in mind that the chemicals tested are intended for widely varying purposes? The extent of the use of animal tests is mainly driven by regulations or by the lack of a suitable in vitro model. Therefore, what considerations are needed for alternative models and how can they be improved so that they can be used as part of the risk assessment process? To address these issues, the European Partnership for Alternative Approaches to Animal Testing (EPAA) working group on prioritization, promotion and implementation of the 3Rs research held a workshop in November, 2008 in Duesseldorf, Germany. Participants included different industry sectors such as pharmaceuticals, cosmetics, industrial- and agro-chemicals. This report describes the outcome of the discussions and recommendations (a) to reduce the number of animals used for determining the ADME properties of chemicals and (b) for considerations and actions regarding in vitro and in silico assays. These included: standardisation and promotion of in vitro assays so that they may become accepted by regulators; increased availability of industry in vivo kinetic data for a central database to increase the power of in silico predictions; expansion of the applicability domains of in vitro and in silico tools (which are not necessarily more applicable or even exclusive to one particular sector) and continued collaborations between regulators, academia and industry. A recommended immediate course of action was to establish an expert panel of users, developers and regulators to define the testing scope of models for different chemical classes. It was agreed by all participants that improvement and harmonization of alternative approaches is needed for all sectors and this will most effectively be achieved by stakeholders from different sectors sharing data.