Risk assessment of parabens in a transcriptomics-based in vitro test.

Parabens have been used for decades as preservatives in food, drugs and cosmetics. The majority however, were banned in 2009 and 2014 leaving only methyl-, ethyl-, propyl-, and butyl-derivates available for subsequent use. Methyl- and propylparaben have been extensively tested in vivo, with no resulting evidence for developmental and reproductive toxicity (DART). In contrast, ethylparaben has not yet been tested for DART in animal experiments, and it is currently debated if additional animal studies are warranted. In order to perform a comparison of the four currently approved parabens, we used a previously established in vitro test based on human induced pluripotent stem cells (iPSC) that are exposed to test substances during their differentiation to neuroectodermal cells. EC50 values for cytotoxicity were 906 µM, 698 µM, 216 µM and 63 µM for methyl-, ethyl-, propyl- and butylparaben, respectively, demonstrating that cytotoxicity increases with increasing alkyl chain length. Genome-wide analysis demonstrated that FDR-adjusted significant gene expression changes occurred only at cytotoxic or close to cytotoxic concentrations, for example 1720 differentially expressed genes (DEG) at 1000 µM ethylparaben, 1 DEG at 316 µM, and no DEG at 100 µM or lower concentrations. The highest concentration of ethylparaben that did not induce any cytotoxicity nor DEG was 1670-fold above the highest concentration reported in biomonitoring studies (60 nM ethylparaben in cord blood). In conclusion, cytotoxicity and gene expression alterations of ethylparaben occurred at concentrations of approximately three orders of magnitude above human blood concentrations; moreover, the substance fitted well into a scenario where toxicity increases with the alkyl chain length, and gene expression changes only occur at cytotoxic or close to cytotoxic concentrations. Therefore, no evidence was obtained suggesting that in vivo DART with ethylparaben would lead to different results as the methyl- or propyl derivates.

Read-across and new approach methodologies applied in a 10-step framework for cosmetics safety assessment - A case study with parabens.

Parabens are esters of para-hydroxybenzoic acid that have been used as preservatives in many types of products for decades including agrochemicals, pharmaceuticals, food and cosmetics. This illustrative case study with propylparaben (PP) demonstrates a 10-step read-across (RAX) framework in practice. It aims at establishing a proof-of-concept for the value added by new approach methodologies (NAMs) in read-across (RAX) for use in a next-generation risk assessment (NGRA) in order to assess consumer safety after exposure to PP-containing cosmetics. In addition to structural and physico-chemical properties, in silico information, toxicogenomics, in vitro toxicodynamic, toxicokinetic data from PBK models, and bioactivity data are used to provide evidence of the chemical and biological similarity of PP and analogues and to establish potency trends for observed effects in vitro. The chemical category under consideration is short (C1-C4) linear chain n-alkyl parabens: methylparaben, ethylparaben, propylparaben and butylparaben. The goal of this case study is to illustrate how a practical framework for RAX can be used to fill a hypothetical data gap for reproductive toxicity of the target chemical PP.

Continuing animal tests on cosmetic ingredients for REACH in the EU

EU cosmetic ingredients are governed by two regulations that conflict. Regulation EC 1223/2009, the Cosmetic Regulation, bans in vivo (animal) testing for cosmetic product safety assessments, including both final products and ingredients. At the same time, the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) regulation can impose in vivo testing of those same ingredients under its chemical testing requirements. Here, we examined REACH dossiers for chemicals for which the only reported use is cosmetics to determine the extent of new in vivo testing caused by REACH. We found the REACH database has 3,206 chemical dossiers with cosmetics as a reported use. Of these, 419 report cosmetics as the only use, and 63 of these have in vivo tests completed after the Cosmetic Regulation ban on in vivo testing. Registrants largely used alternative, non-animal methods to evaluate ingredients for REACH, but some still conducted new in vivo tests to comply with REACH requirements for toxicity data and worker safety assessments. In some cases, ECHA, the agency that evaluates REACH dossiers, rejected registrants' alternative methods as insufficient and required new in vivo tests. As ECHA continues to evaluate dossiers, more requests for in vivo tests are likely. REACH tests on cosmetic ingredients appear only as "industrial chemicals legislation" tests in EU reports. Given the importance to consumers and the cosmetic industry of having cosmetics free of animal testing, the public should be made aware of REACH testing until the conflict between the regulations is resolved.

Application of grouping and read-across for the evaluation of parabens of different chain lengths with a particular focus on endocrine properties.

This article presents the outcomes of higher-tier repeated-dose toxicity studies and developmental and reproductive toxicity (DART) studies using Wistar rats requested for methyl paraben and propyl paraben under the European Union chemicals legislation. All studies revealed no-observed adverse effects (NOAELs) at 1000 mg/kg body weight/day. These findings (absence of effects) were then used to interpolate the hazard profile for ethyl paraben, further considering available data for butyl paraben. The underlying read-across hypothesis (all shorter-chained linear n-alkyl parabens are a 'category' based on very high structural similarity and are transformed to a common compound) was confirmed by similarity calculations and comparative in vivo toxicokinetics screening studies for methyl paraben, ethyl paraben, propyl paraben and butyl paraben. All four parabens were rapidly taken up systemically following oral gavage administration to rats, metabolised to p-hydroxybenzoic acid, and rapidly eliminated (parabens within one hour; p-hydroxybenzoic acid within 4-8 h). Accordingly, for ethyl paraben, the NOAELs for repeated-dose toxicity and DART were interpolated to be 1000 mg/kg body weight/day. Finally, all evidence was evaluated to address concerns expressed in the literature that parabens might be endocrine disruptors. This evaluation showed that the higher-tier studies do not provide any indication for any endocrine disrupting property. This is the first time that a comprehensive dataset from higher-tier in vivo studies following internationally agreed test protocols has become available for shorter-chained linear n-alkyl parabens. Consistently, the dataset shows that these parabens are devoid of repeated-dose toxicity and do not possess any DART or endocrine disrupting properties.

The Grouping and Assessment Strategy for Organic Pigments (GRAPE): Scientific evidence to facilitate regulatory decision-making.

This article presents the Grouping and Assessment Strategy for Organic Pigments (GRAPE). GRAPE is driven by the hypotheses that low (bio)dissolution and low permeability indicate absence of systemic bioavailability and hence no systemic toxicity potential upon oral exposure, and, for inhalation exposure, that low (bio)dissolution (and absence of surface reactivity, dispersibility and in vitro effects) indicate that the organic pigment is a 'poorly soluble particle without intrinsic toxicity potential'. In GRAPE Tier 1, (bio)solubility and (bio)dissolution are assessed, and in Tier 2, in vitro Caco-2 permeability and in vitro alveolar macrophage activation. Thereafter, organic pigments are grouped by common properties (further considering structural similarity depending on the regulatory requirements). In Tier 3, absence of systemic bioavailability is verified by limited in vivo screening (rat 28-day oral and 5-day inhalation toxicity studies). If Tier 3 confirms no (or only very low) systemic bioavailability, all higher-tier endpoint-specific animal testing is scientifically not-relevant. Application of the GRAPE can serve to reduce animal testing needs for all but few representative organic pigments within a group. GRAPE stands in line with the EU REACH Regulation (Registration, Evaluation, Authorisation and Restriction of Chemicals). An ongoing research project aims at establishing a proof-of-concept of the GRAPE.

Prediction of human drug-induced liver injury (DILI) in relation to oral doses and blood concentrations.

Drug-induced liver injury (DILI) cannot be accurately predicted by animal models. In addition, currently available in vitro methods do not allow for the estimation of hepatotoxic doses or the determination of an acceptable daily intake (ADI). To overcome this limitation, an in vitro/in silico method was established that predicts the risk of human DILI in relation to oral doses and blood concentrations. This method can be used to estimate DILI risk if the maximal blood concentration (Cmax) of the test compound is known. Moreover, an ADI can be estimated even for compounds without information on blood concentrations. To systematically optimize the in vitro system, two novel test performance metrics were introduced, the toxicity separation index (TSI) which quantifies how well a test differentiates between hepatotoxic and non-hepatotoxic compounds, and the toxicity estimation index (TEI) which measures how well hepatotoxic blood concentrations in vivo can be estimated. In vitro test performance was optimized for a training set of 28 compounds, based on TSI and TEI, demonstrating that (1) concentrations where cytotoxicity first becomes evident in vitro (EC10) yielded better metrics than higher toxicity thresholds (EC50); (2) compound incubation for 48 h was better than 24 h, with no further improvement of TSI after 7 days incubation; (3) metrics were moderately improved by adding gene expression to the test battery; (4) evaluation of pharmacokinetic parameters demonstrated that total blood compound concentrations and the 95%-population-based percentile of Cmax were best suited to estimate human toxicity. With a support vector machine-based classifier, using EC10 and Cmax as variables, the cross-validated sensitivity, specificity and accuracy for hepatotoxicity prediction were 100, 88 and 93%, respectively. Concentrations in the culture medium allowed extrapolation to blood concentrations in vivo that are associated with a specific probability of hepatotoxicity and the corresponding oral doses were obtained by reverse modeling. Application of this in vitro/in silico method to the rat hepatotoxicant pulegone resulted in an ADI that was similar to values previously established based on animal experiments. In conclusion, the proposed method links oral doses and blood concentrations of test compounds to the probability of hepatotoxicity.

Toxicity testing of poorly soluble particles, lung overload and lung cancer.

In 2013, an ECETOC Task Force evaluated scientific understanding of the 'lung overload' hypothesis. As there is no evidence that humans develop lung tumours following exposure to poorly soluble particles (PSPs), emphasis was given to the observed higher sensitivity and specificity of rat lung responses and potential impacts of this on human risk assessment. Key arguments and outcomes are summarised here, together with discussion of additional findings published since 2013. Inhalation exposure to PSPs in all species is associated with localised pulmonary toxicity initiated by a persistent pro-inflammatory response to particle deposition. Events in the rat indicate a plausible adverse outcome pathway for lung tumour development following exposure to PSPs under overload conditions. A different particle lung translocation pattern compared to rats make humans less sensitive to developing comparable lung overload conditions and appears to also preclude tumour formation, even under severe and prolonged exposure conditions. Evidence continues to suggest that the rat lung model is unreliable as a predictor for human lung cancer risk. However, it is a sensitive model for detecting various thresholded inflammatory markers, with utility for non-neoplastic risk assessment purposes. It is noteworthy that preventing inflammatory rat lung responses will also inhibit development of neoplastic outcomes.

In chemico, in vitro and in vivo comparison of the skin sensitizing potential of eight unsaturated and one saturated lipid compounds.

The applicability of the Direct Peptide Reactivity Assay (DPRA), the KeratinoSens™ assay and the human cell line activation test (OECD Test Guidelines 442C, 442D, 442E) in predicting the skin sensitising potential of nine lipid (bio)chemicals was investigated. The results from the three assays were integrated using a published prediction model (PM), by which skin sensitisation is predicted if at least two of the three assays yield positive results. Of the eight test substances that were classified as non-sensitisers using available Guinea Pig Maximisation Test (GPMT) data, only five were correctly predicted as 'negative' in the PM. (However, only two were correctly predicted as 'negative' in the murine Local Lymph Node Assay.) The one lipid (bio)chemical that tested positive in the GPMT was also positive applying the PM. Based upon the outcome of the present study, lipid (bio)chemicals with a log Kow up to 7-8 appear amenable to the three assays. However, solubility problems, that were not evident initially, affected the performance of the DPRA. Further investigations are merited to address the conclusiveness of negative test results with concurrent lack of cytotoxicity in the in vitro assays, to evaluate if poorly soluble substances come into contact with the cells.

Case studies putting the decision-making framework for the grouping and testing of nanomaterials (DF4nanoGrouping) into practice.

Case studies covering carbonaceous nanomaterials, metal oxide and metal sulphate nanomaterials, amorphous silica and organic pigments were performed to assess the Decision-making framework for the grouping and testing of nanomaterials (DF4nanoGrouping). The usefulness of the DF4nanoGrouping for nanomaterial hazard assessment was confirmed. In two tiers that rely exclusively on non-animal test methods followed by a third tier, if necessary, in which data from rat short-term inhalation studies are evaluated, nanomaterials are assigned to one of four main groups (MGs). The DF4nanoGrouping proved efficient in sorting out nanomaterials that could undergo hazard assessment without further testing. These are soluble nanomaterials (MG1) whose further hazard assessment should rely on read-across to the dissolved materials, high aspect-ratio nanomaterials (MG2) which could be assessed according to their potential fibre toxicity and passive nanomaterials (MG3) that only elicit effects under pulmonary overload conditions. Thereby, the DF4nanoGrouping allows identifying active nanomaterials (MG4) that merit in-depth investigations, and it provides a solid rationale for their sub-grouping to specify the further information needs. Finally, the evaluated case study materials may be used as source nanomaterials in future read-across applications. Overall, the DF4nanoGrouping is a hazard assessment strategy that strictly uses animals as a last resort.

A decision-making framework for the grouping and testing of nanomaterials (DF4nanoGrouping).

The European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) 'Nano Task Force' proposes a Decision-making framework for the grouping and testing of nanomaterials (DF4nanoGrouping) that consists of 3 tiers to assign nanomaterials to 4 main groups, to perform sub-grouping within the main groups and to determine and refine specific information needs. The DF4nanoGrouping covers all relevant aspects of a nanomaterial's life cycle and biological pathways, i.e. intrinsic material and system-dependent properties, biopersistence, uptake and biodistribution, cellular and apical toxic effects. Use (including manufacture), release and route of exposure are applied as 'qualifiers' within the DF4nanoGrouping to determine if, e.g. nanomaterials cannot be released from a product matrix, which may justify the waiving of testing. The four main groups encompass (1) soluble nanomaterials, (2) biopersistent high aspect ratio nanomaterials, (3) passive nanomaterials, and (4) active nanomaterials. The DF4nanoGrouping aims to group nanomaterials by their specific mode-of-action that results in an apical toxic effect. This is eventually directed by a nanomaterial's intrinsic properties. However, since the exact correlation of intrinsic material properties and apical toxic effect is not yet established, the DF4nanoGrouping uses the 'functionality' of nanomaterials for grouping rather than relying on intrinsic material properties alone. Such functionalities include system-dependent material properties (such as dissolution rate in biologically relevant media), bio-physical interactions, in vitro effects and release and exposure. The DF4nanoGrouping is a hazard and risk assessment tool that applies modern toxicology and contributes to the sustainable development of nanotechnological products. It ensures that no studies are performed that do not provide crucial data and therefore saves animals and resources.

A critical appraisal of existing concepts for the grouping of nanomaterials.

The grouping of substances serves to streamline testing for regulatory purposes. General grouping approaches for chemicals have been implemented in, e.g., the EU chemicals regulation. While specific regulatory frameworks for the grouping of nanomaterials are unavailable, this topic is addressed in different publications, and preliminary guidance is provided in the context of substance-related legislation or the occupational setting. The European Centre for Ecotoxicology and Toxicology of Chemicals Task Force on the Grouping of Nanomaterials reviewed available concepts for the grouping of nanomaterials for human health risk assessment. In their broad conceptual design, the evaluated approaches are consistent or complement each other. All go beyond the determination of mere structure-activity relationships and are founded on different aspects of the nanomaterial life cycle. These include the NM's material properties and biophysical interactions, specific types of use and exposure, uptake and kinetics, and possible early and apical biological effects. None of the evaluated grouping concepts fully take into account all of these aspects. Subsequent work of the Task Force will aim at combining the available concepts into a comprehensive 'multiple perspective' framework for the grouping of nanomaterials that will address all of the mentioned aspects of their life cycles.

Evaluating the sensitization potential of surfactants: integrating data from the local lymph node assay, guinea pig maximization test, and in vitro methods in a weight-of-evidence approach.

An integral part of hazard and safety assessments is the estimation of a chemical's potential to cause skin sensitization. Currently, only animal tests (OECD 406 and 429) are accepted in a regulatory context. Nonanimal test methods are being developed and formally validated. In order to gain more insight into the responses induced by eight exemplary surfactants, a battery of in vivo and in vitro tests were conducted using the same batch of chemicals. In general, the surfactants were negative in the GPMT, KeratinoSens and hCLAT assays and none formed covalent adducts with test peptides. In contrast, all but one was positive in the LLNA. Most were rated as being irritants by the EpiSkin assay with the additional endpoint, IL1-alpha. The weight of evidence based on this comprehensive testing indicates that, with one exception, they are non-sensitizing skin irritants, confirming that the LLNA tends to overestimate the sensitization potential of surfactants. As results obtained from LLNAs are considered as the gold standard for the development of new nonanimal alternative test methods, results such as these highlight the necessity to carefully evaluate the applicability domains of test methods in order to develop reliable nonanimal alternative testing strategies for sensitization testing.

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

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

Comparative testing for the identification of skin-sensitizing potentials of nonionic sugar lipid surfactants.

The Local Lymph Node Assay (LLNA) is the preferred test for the identification of skin-sensitizing potentials of chemicals in Europe and is also the first choice method within REACH. In the formal validation, only a very few surfactant chemicals were evaluated and SDS was identified as a false positive. In this study, 10 nonionic sugar lipid surfactants were tested in an LLNA, guinea pig maximization test (GPMT) and human repeated insult patch test. Of the 10 surfactants tested in the LLNA, 5 showed stimulation indices above 3.0. Three of five positive reactions were concomitant with signs of skin irritation indicated by an increase in ear thickness. In the GPMT, all test products were classified as nonsensitizers. In human volunteers, no skin reactions suggestive of sensitization were reported. In conclusion, these results are indicative of the LLNA overestimating sensitization potentials for this category of chemicals. This may in part be due to irritant effects generated by these surfactants. Until suitable nonanimal alternative tests obtain regulatory acceptance, use of other tests, e.g. GPMTs, may in cases be justified. Results such as these need be taken into account when developing nonanimal alternative methods to ensure reliable data sets for method validation purposes.

Application of a weight of evidence approach to assessing discordant sensitisation datasets: implications for REACH.

The local lymph node assay (LLNA) is the assay of choice in European regulatory toxicology. As with other toxicology/sensitisation assays, it has a potential for false results, the anionic surfactant sodium lauryl sulphate (SLS) representing a classic example. In the work reported here, examples of false positives in the LLNA are compared to published "benchmarks" such as SLS. Clear false positives (e.g. oleic acid) are also contrasted with examples where data interpretation is more challenging. As the LLNA will be applicable to >30,000 chemicals under REACH, and in the light of animal welfare considerations to do no more than the absolute minimum of animal testing, results from a single LLNA often represent the only available data on sensitisation. This reinforces the need to ensure data from this assay are interpreted intelligently, using scientific analysis of results and considering the weight of evidence, before decisions are made on which substances should be classified as representing a skin sensitisation hazard. In chemical classes where the LLNA has been shown to be an inappropriate assay other standardised methods (e.g. the Buehler or Magnusson and Kligman guinea pig tests [OECD 406]) should be employed as the first choice assays.