Gamze Ates

Screening of repeated dose toxicity data present in SCC(NF)P/SCCS safety evaluations of cosmetic ingredients

Alternative methods, replacing animal testing, are urgently needed in view of the European regulatory changes in the field of cosmetic products and their ingredients. In this context, a joint research initiative called SEURAT was recently raised by the European Commission and COLIPA, representing the European cosmetics industry, with the overall goal of developing an animal-free repeated dose toxicity testing strategy for human safety assessment purposes. Although cosmetic ingredients are usually harmless for the consumer, one of the initial tasks of this research consortium included the identification of organs that could potentially be affected by cosmetic ingredients upon systemic exposure. The strategy that was followed hereof is described in the present paper and relies on the systematic evaluation, by using a self-generated electronic databank, of published reports issued by the scientific committee of DG SANCO responsible for the safety of cosmetic ingredients. By screening of the repeated dose toxicity studies present in these reports, it was found that the liver is potentially the most frequently targeted organ by cosmetic ingredients when orally administered to experimental animals, followed by the kidney and the spleen. Combined listing of altered morphological, histopathological, and biochemical parameters subsequently indicated the possible occurrence of hepatotoxicity, including steatosis and cholestasis, triggered by a limited number of cosmetic compounds. These findings are not only of relevance for the in vitro modeling efforts and choice of compounds to be tested in the SEURAT project cluster, but also demonstrate the importance of using previously generated toxicological data through an electronic databank for addressing specific questions regarding the safety evaluation of cosmetic ingredients.

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.

Way forward in case of a false positive in vitro genotoxicity result for a cosmetic substance

The currently used regulatory in vitro mutagenicity/genotoxicity test battery has a high sensitivity for detecting genotoxicants, but it suffers from a large number of irrelevant positive results (i.e. low specificity) thereby imposing the need for additional follow-up by in vitro and/or in vivo genotoxicity tests. This could have a major impact on the cosmetic industry in Europe, seen the imposed animal testing and marketing bans on cosmetics and their ingredients. Afflicted, but safe substances could therefore be lost. Using the example of triclosan, a cosmetic preservative, we describe here the potential applicability of a human toxicogenomics-based in vitro assay as a potential mechanistically based follow-up test for positive in vitro genotoxicity results. Triclosan shows a positive in vitro chromosomal aberration test, but is negative during in vivo follow-up tests. Toxicogenomics analysis unequivocally shows that triclosan is identified as a compound acting through non-DNA reactive mechanisms. This proof-of-principle study illustrates the potential of genome-wide transcriptomics data in combination with in vitro experimentation as a possible weight-of-evidence follow-up approach for de-risking a positive outcome in a standard mutagenicity/genotoxicity battery. As such a substantial number of cosmetic compounds wrongly identified as genotoxicants could be saved for the future.

In silico tools and transcriptomics analyses in the mutagenicity assessment of cosmetic ingredients: a proof-of-principle on how to add weight to the evidence

Prior to the downstream development of chemical substances, including pharmaceuticals and cosmetics, their influence on the genetic apparatus has to be tested. Several in vitro and in vivo assays have been developed to test for genotoxicity. In a first tier, a battery of two to three in vitro tests is recommended to cover mutagenicity, clastogenicity and aneugenicity as main endpoints. This regulatory in vitro test battery is known to have a high sensitivity, which is at the expense of the specificity. The high number of false positive in vitro results leads to excessive in vivo follow-up studies. In the case of cosmetics it may even induce the ban of the particular compound since in Europe the use of experimental animals is no longer allowed for cosmetics. In this article, an alternative approach to derisk a misleading positive Ames test is explored. Hereto we first tested the performance of five existing computational tools to predict the potential mutagenicity of a data set of 132 cosmetic compounds with a known genotoxicity profile. Furthermore, we present, as a proof-of-principle, a strategy in which a combination of computational tools and mechanistic information derived from in vitro transcriptomics analyses is used to derisk a misleading positive Ames test result. Our data shows that this strategy may represent a valuable tool in a weight-of-evidence approach to further evaluate a positive outcome in an Ames test.

Testing chemical carcinogenicity by using a transcriptomics HepaRG-based model?

The EU FP6 project carcinoGENOMICS explored the combination of toxicogenomics and in vitro cell culture models for identifying organotypical genotoxic- and non-genotoxic carcinogen-specific gene signatures. Here the performance of its gene classifier, derived from exposure of metabolically competent human HepaRG cells to prototypical non-carcinogens (10 compounds) and hepatocarcinogens (20 compounds), is reported. Analysis of the data at the gene and the pathway level by using independent biostatistical approaches showed a distinct separation of genotoxic from non-genotoxic hepatocarcinogens and non-carcinogens (up to 88 % correct prediction). The most characteristic pathway responding to genotoxic exposure was DNA damage. Interlaboratory reproducibility was assessed by blindly testing of three compounds, from the set of 30 compounds, by three independent laboratories. Subsequent classification of these compounds resulted in correct prediction of the genotoxicants. As expected, results on the non-genotoxic carcinogens and the non-carcinogens were less predictive. In conclusion, the combination of transcriptomics with the HepaRG in vitro cell model provides a potential weight of evidence approach for the evaluation of the genotoxic potential of chemical substances.

A novel genotoxin-specific qPCR array based on the metabolically competent human HepaRG™ cell line as a rapid and reliable tool for improved in vitro hazard assessment

Although the value of the regulatory accepted batteries for in vitro genotoxicity testing is recognized, they result in a high number of false positives. This has a major impact on society and industries developing novel compounds for pharmaceutical, chemical, and consumer products, as afflicted compounds have to be (prematurely) abandoned or further tested on animals. Using the metabolically competent human HepaRG™ cell line and toxicogenomics approaches, we have developed an upgraded, innovative, and proprietary gene classifier. This gene classifier is based on transcriptomic changes induced by 12 genotoxic and 12 non-genotoxic reference compounds tested at sub-cytotoxic concentrations, i.e., IC10 concentrations as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The resulting gene classifier was translated into an easy-to-handle qPCR array that, as shown by pathway analysis, covers several different cellular processes related to genotoxicity. To further assess the predictivity of the tool, a set of 5 known positive and 5 known negative test compounds for genotoxicity was evaluated. In addition, 2 compounds with debatable genotoxicity data were tested to explore how the qPCR array would classify these. With an accuracy of 100%, when equivocal results were considered positive, the results showed that combining HepaRG™ cells with a genotoxin-specific qPCR array can improve (geno)toxicological hazard assessment. In addition, the developed qPCR array was able to provide additional information on compounds for which so far debatable genotoxicity data are available. The results indicate that the new in vitro tool can improve human safety assessment of chemicals in general by basing predictions on mechanistic toxicogenomics information.

The Vitotox and ToxTracker assays: A two-test combination for quick and reliable assessment of genotoxic hazards

To ensure safety for humans, it is essential to characterize the genotoxic potential of new chemical entities, such as pharmaceutical and cosmetic substances. In a first tier, a battery of in vitro tests is recommended by international regulatory agencies. However, these tests suffer from inadequate specificity: compounds may be wrongly categorized as genotoxic, resulting in unnecessary, time-consuming, and expensive in vivo follow-up testing. In the last decade, novel assays (notably, reporter-based assays) have been developed in an attempt to overcome these drawbacks. Here, we have investigated the performance of two in vitro reporter-based assays, Vitotox and ToxTracker. A set of reference compounds was selected to span a variety of mechanisms of genotoxic action and applicability domains (e.g., pharmaceutical and cosmetic ingredients). Combining the performance of the two assays, we achieved 93% sensitivity and 79% specificity for prediction of gentoxicity for this set of compounds. Both assays permit quick high-throughput analysis of drug candidates, while requiring only small quantities of the test substances. Our study shows that these two assays, when combined, can be a reliable method for assessment of genotoxicity hazard.

Genetic deletion of xCT attenuates peripheral and central inflammation and mitigates LPS-induced sickness and depressive-like behavior in mice

The communication between the immune and central nervous system (CNS) is affected in many neurological disorders. Peripheral injections of the endotoxin lipopolysaccharide (LPS) are widely used to study this communication: an LPS challenge leads to a biphasic syndrome that starts with acute sickness and is followed by persistent brain inflammation and chronic behavioral alterations such as depressive‐like symptoms. In vitro, the response to LPS treatment has been shown to involve enhanced expression of system xc− . This cystine‐glutamate antiporter, with xCT as specific subunit, represents the main glial provider of extracellular glutamate in mouse hippocampus. Here we injected male xCT knockout and wildtype mice with a single intraperitoneal dose of 5 mg/kg LPS. LPS‐injection increased hippocampal xCT expression but did not alter the mainly astroglial localization of the xCT protein. Peripheral and central inflammation (as defined by cytokine levels and morphological activation of microglia) as well as LPS‐induced sickness and depressive‐like behavior were significantly attenuated in xCT‐deficient mice compared with wildtype mice. Our study is the first to demonstrate the involvement of system xc− in peripheral and central inflammation in vivo and the potential therapeutic relevance of its inhibition in brain disorders characterized by peripheral and central inflammation, such as depression.

Assaying Cellular Viability Using the Neutral Red Uptake Assay

The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cationic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation. The neutral red uptake assay is mainly used for hazard assessment in in vitro toxicology applications. This method has also been introduced in regulatory recommendations as part of 3T3-NRU-phototoxicity-assay, which was regulatory accepted in all EU member states in 2000 and in the OECD member states in 2004 as a test guideline (TG 432). The present protocol describes the neutral red uptake assay using the human hepatoma cell line HepG2, which is often employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetyl salicylic acid is assessed.

Contents Vol. 29, 2016

N. Ahmad, Madison, Wis. C. Antoniou, Athens J.M. Baron, Aachen E. Benfeldt, Roskilde E. Berardesca, Rome K. De Paepe, Brussels P. Elsner, Jena A. Farkas, Zurich N. Garcia Bartels, Berlin M.W. Greaves, London R.H. Guy, Bath S. Hedtrich, Berlin E.M. Jackson, Bonney Lake, Wash. H. Kandárová, Ashland, Mass. L. Kemeny, Szeged J. Kresken, Viersen J. Krutmann, Düsseldorf B. Lange-Asschenfeldt, Klagenfurt am Wörthersee R. Neubert, Halle T. Ruzicka, Munich M. Schäfer-Korting, Berlin M. Schmuth, Innsbruck S. Seidenari, Modena P.W. Wertz, Iowa City, Iowa J. Wohlrab, Halle L. Zastrow, Monaco Journal of Pharmacological and Biophysical Research