European Centre For The Validation Of Alternative Methods Research Articles

Alternative in vitro models used in the main safety tests of cosmetic products and new challenges.

Guided by ethical considerations and regulatory requirements such as the 7th Amendment to the European Cosmetics Directive N° 1223/2009, the cosmetic industry has developed and evaluated alternative test strategies such as in vitro assays, in silico approaches for toxicological endpoints and efficacy of cosmetic products and cosmetics ingredients. In consequence, the European Centre for the Validation of Alternative Methods (ECVAM) has proposed a list of validated cell-based in vitro models for predicting the safety and toxicity of cosmetic ingredients. These models have been demonstrated as valuable and effective tools to overcome the limitations of animal in vivo studies. For example, 3D human skin equivalent models are used to evaluate skin irritation potential; and excised human skin is used as the gold standard for the evaluation of dermal absorption. This review presents, in relation to the regulatory requirements, the main alternative in vitro models used in the safety tests of cosmetic products, focusing on skin sensitization, skin corrosion, skin irritation and skin absorption, with advantages and limitations of each model. Recent innovative 3D cell technologies such as Organ-on-a-Chip (OoC) models that can bring significant improvements for toxicology and efficacy testing are also presented. The development of OoC technology is promising for assessing the toxicity of substances contained in cosmetics, particularly for repeated dose toxicity, for which no alternative in vitro methods are currently available. Nevertheless, aside from the challenges, the technology needs to be validated and accepted by regulatory organizations as an effective method. Collaboration between researchers, regulatory organizations and industry would be required to achieve this validation.

Dynamically monitoring cellular γ-H2AX reveals the potential of carcinogenicity evaluation for genotoxic compounds.

Amongst all toxicological endpoints, carcinogenicity might pose the greatest concern. Genetic damage has been considered an important underlying mechanism for the carcinogenicity of chemical substances. The demand for in vitro genotoxic tests as alternative approaches is growing rapidly with the implementation of new regulations for compounds. However, currently available in vitro genotoxicity tests are often limited by relatively high false positive rates. Moreover, few studies have explored carcinogenicity potential by in vitro genotoxicity testing due to the shortage of suitable toxicological biomarkers to link gene damage with cancer risk. γ-H2AX is a recently acknowledged attractive endpoint (biomarker) for evaluating DNA damage and can simultaneously reflect the DNA damage response and repair of cells. We previously reported an ultrasensitive and reliable method, namely stable-isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS), for detecting cellular γ-H2AX and evaluating genotoxic chemicals. More importantly, our method can dynamically monitor the specific processes of genotoxic compounds affecting DNA damage and repair reflected by the amount of γ-H2AX. To clarify the possibility of using this method to assess the potential carcinogenicity of genotoxic chemicals, we applied it to a set of 69 model compounds recommended by the European Center for the Validation of Alternative Methods (ECVAM), with already-characterized genotoxic potential. Compared to conventional in vitro genotoxicity assays, including the Ames test, the γ-H2AX assay by MS has high accuracy (94-96%) due to high sensitivity and specificity (88% and 100%, respectively). The dynamic profiles of model compounds after exposure in HepG2 cells were explored, and a mathematical approach was employed to simulate and quantitatively model the DNA repair kinetics of genotoxic carcinogens (GCs) based on γ-H2AX time-effect curves up to 8h. Two crucial parameters, i.e., k (rate of γ-H2AX decay) and t50 (time required for γ-H2AX from maximum decrease to half) estimated by the least squares method, were achieved. An open web server to help researchers calculate these two key parameters and profile simulated curves of the tested compound is available online ( http://ccb1.bmi.ac.cn:81/shiny-server/sample-apps/prediction1/ ). We detected a positive association between carcinogenic levels and k and t50 values of γ-H2AX in tested GCs, validating the potential of using this MS-based γ-H2AX in vitro assay to help preliminarily evaluate carcinogenicity and assess genotoxicity. This approach may be used alone or integrated into an existing battery of in vitro genetic toxicity tests.

Pre-validation study of alternative developmental toxicity test using mouse embryonic stem cell-derived embryoid bodies

The embryoid body test (EBT) is a developmental toxicity test method that assesses the half inhibitory concentrations of substances in the area of embryoid bodies (EBs), and in the viability of mouse embryonic stem cells (ESCs) and fibroblasts (3T3 cells) following chemical exposure for three and four days, respectively. In the previous study, the EBT showed more advanced than the embryonic stem cell test (EST) from the European Centre for the Validation of Alternative Methods (ECVAM) applying cardiac differentiation of mouse ESCs, because the EBT greatly reduced the exposure time, labor, and amount of materials required, and misclassification of embryotoxic potential. This pre-validation study evaluated the predictive accuracy of the EBT using 26 coded test substances by two steps: intra-laboratory and inter-laboratory reproducibility tests. Since some substances have different embryotoxic potentials at different pregnancy periods, in this study, a new prediction model consisting of non-toxic and toxic classes was used, instead of the existing prediction model assessing embryotoxicants in four classes. The results of the intra- and inter-laboratory tests were highly accurate (above 80%) when substances were classified using the predictive model. In conclusion, EBT can accurately classify various embryotoxicants in a short time with less effort and greater validation.

Electrical activity of developing cardiomyocytes

Event Abstract Back to Event Electrical activity of developing cardiomyocytes Saskia Wuttke1*, Gabriele Brockerhoff1, Laura Nimtz1, Julia Tigges1 and Ellen Fritsche1 1 Leibniz-Institut für umweltmedizinische Forschung (IUF), Modern Risk Assessment and Sphere Biology, Germany Today developmental toxicity testing is often performed on animals due to several guidelines (OECD, U.S. EPA , ). This method is time and resource-intensive resulting in the need of alternative methods. The European Centre for the Validation of Alternative Methods (ECVAM) is supporting alternative methods and validated the mouse embryonic stem cell test (mEST) as an appropriate test for developmental toxicity testing . The mEST is based on a permanent cell line of mouse embryonic stem cells that are differentiated into cardiomyocytes. The quantity of beating cardiomyocytes is the respective readout . The extrapolation from mouse to human is discussed controversially . Therefore, we establish a human embryonic stem cell test (hEST). As the usage of human embryos has ethical concerns, we use human induced pluripotent stem cells (hiPSC). These cells originate from fibroblasts which were reprogrammed into stem cells that cover all the characteristics of embryonic stem cells like self-renewal and pluripotency . To establish a hEST, hiPSC are differentiated into cardiomyocytes with the aim of evaluating more endpoints than the mEST. Therefore, we already established the differentiation of hiPSC to cardiomyocytes, achieving beating cardiomyocytes evenly in the entire dish which is a basic requirement for recording the electrical activity on microelectrode arrays (MEA). As the differentiation protocol represents the embryonic development of cardiomyocytes, the day of the first electrical signal could be a specific functional endpoint to measure. Furthermore, the number of electrodes recording a signal over time could be evaluated as another endpoint indicating maturation of the cells. When the differentiation of hiPSC to cardiomyocytes is established on MEAs, it is possible to screen substances for their toxic potential on the electrical activity of cardiomyocytes and therefore on the entire human cardiac system. References 1 Test No. 414 Prenatal Development Toxicity Study: TG414 (OECD Publishing, 2001). 2 Test No. 426 Developmental Neurotoxicity Study: TG426 (OECD Publishing, 2007). 3 A. E. M. Seiler and H. Spielmann, ‘The validated embryonic stem cell test to predict embryotoxicity in vitro’, Nature protocols 6 (2011), 961–78. 4 H.-y. Lee, A. L. Inselman, J. Kanungo and D. K. Hansen, ‘Alternative models in developmental toxicology’, Systems biology in reproductive medicine 58 (2012), 10–22. 5 J. Bailey, M. Thew and M. Balls, ‘Predicting human drug toxicity and safety via animal tests: Can any one species predict drug toxicity in any other, and do monkeys help?’, Alternatives to laboratory animals : ATLA 43 (2015), 393–403. 6 K. Takahashi, K. Tanabe, M. Ohnuki, M. Narita, T. Ichisaka, K. Tomoda and S. Yamanaka, ‘Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors’, Cell 131 (2007), 861–72. Keywords: hiPSC, cardiomyocytes, MEA, embryonic, development Conference: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018. Presentation Type: Poster Presentation Topic: Stem cell-derived applications Citation: Wuttke S, Brockerhoff G, Nimtz L, Tigges J and Fritsche E (2019). Electrical activity of developing cardiomyocytes. Conference Abstract: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays. doi: 10.3389/conf.fncel.2018.38.00012 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 17 Mar 2018; Published Online: 17 Jan 2019. * Correspondence: Ms. Saskia Wuttke, Leibniz-Institut für umweltmedizinische Forschung (IUF), Modern Risk Assessment and Sphere Biology, Dusseldorf, NRW, 40225, Germany, saskia.wuttke@iuf-duesseldorf.de Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Saskia Wuttke Gabriele Brockerhoff Laura Nimtz Julia Tigges Ellen Fritsche Google Saskia Wuttke Gabriele Brockerhoff Laura Nimtz Julia Tigges Ellen Fritsche Google Scholar Saskia Wuttke Gabriele Brockerhoff Laura Nimtz Julia Tigges Ellen Fritsche PubMed Saskia Wuttke Gabriele Brockerhoff Laura Nimtz Julia Tigges Ellen Fritsche Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

The embryonic toxicity evaluation of deoxynivalenol (DON) by murine embryonic stem cell test and human embryonic stem cell test models

Deoxynivalenol (DON) is a group B trichothecene and a common contaminant of food crops worldwide. DON is known to cause a spectrum of diseases in animals and humans such as vomiting and gastroenteritis and has been shown to cross the human placental barrier; therefore, dietary exposure during pregnancy could lead to exposure of the fetus. Since the mechanism of DON toxicity action has not been thoroughly elucidated, further evaluation of the underlying mechanisms of DON's embryotoxicity is needed. This is especially important for developing exposure guidance recommendations, especially those targeted towards pregnant women.In the present study, murine embryonic stem cell test (mEST) and human embryonic stem cell test (hEST) models were developed according to protocols of the European Centre for the Validation of Alternative Methods (ECVAM). Different concentrations of DON were administered to mouse embryonic stem cells D3 (mESC-D3), mouse Balb/c-3T3 (3T3) embryo fibroblast cells, and human embryonic stem cells H9 (hESC-H9) for 10 days to detect the 50% inhibitory proliferation concentration (IC50) of mESC-D3 cells, 3T3 cells, and hESC-H9 cells with DON. Differentiation of ESCs was initiated by embryoid body (EBs) formation. EBs were exposed to different concentrations of DONfor 10 days. The expression of cardiomyocyte differentiation gene alpha-myosin heavy chain (α-MHC) was detected by real-time PCR and the 50% inhibition of cardiomyocyte differentiation (ID50) was determined. Based on the values of IC50 and ID50, functions I, II, and III were calculated by three linear discriminant functions in the EST model and the embryotoxicity of DON was described by comparing the three functions. Results of the three endpoints of DON in murine EST were 0.141 μg/ml (IC50 3T3), 0.085 μg/ml (IC50 D3), and 0.110 μg/ml (ID50 D3). The function I (−34.43), function Ⅱ (−18.62), and function III (1.98) were calculated for DON by themEST model. The three endpoints of DON in hEST were 0.13 μg/ml (IC50 3T3), 0.11 μg/ml (IC50 H9), and 0.078 μg/ml (ID50). Function I (−25.97), function Ⅱ (−13.18), and function III (−0.12) were calculated based on IC50 and ID50. Since function III > function II > function I, according to the EST criteria, DON was determined to have strong embryo toxicity both by mEST and hEST. Moreover, the hEST model, which excluded species differences, is suggested to be a more accurate and reliable method for the evaluation of chemical embryotoxicity.

Construction of Three-Dimensional Dermo-Epidermal Skin Equivalents Using Cell Coating Technology and Their Utilization as Alternative Skin for Permeation Studies and Skin Irritation Tests.

In vitro generated human skin equivalents are generating interest as promising tools in basic study, as alternatives to animal testing, and for clinical applications in regenerative medicine. For prediction of skin irritation and corrosion, three-dimensional human skin equivalents consisting of differentiated human keratinocytes (KCs) have been developed and some models have been internationally accepted. However, more delicate assessments using full-thickness skin models, such as skin sensitization tests, cannot be performed due to the lack of a dermis containing fibroblasts or appendages. In a previous study, we developed dermo-epidermal human skin equivalents (DESEs) using a cell coating technique, which employs cell surface coating by layer-by-layer assembled extracellular matrix (ECM) films. The DESEs with dermis consisting of normal human dermal fibroblasts (NHDFs) and epidermis consisting of human KCs were easily fabricated by using this technology. In this study, the constructed DESEs were evaluated as an alternative skin for skin permeation and irritation tests. A good relationship of permeability coefficient of chemicals was observed between the DESEs and human skin data. We investigated whether the DESEs, a new in vitro skin model, are capable of identifying skin irritant and nonirritant substances among 20 reference chemicals. It was confirmed that the DESEs are applicable to skin irritation testing as defined in the European Centre for the Validation of Alternative Methods (ECVAM) Performance Standard (OECD Test Guideline 439). We further studied the construction of DESEs with density-controlled blood capillary networks using human umbilical vein endothelial cells (HUVECs). The results suggest that DESEs allowing incorporation of skin appendages are more promising alternatives to animal testing and can be applied to the design of physiologically relevant in vitro skin models.

Comparison of embryotoxicity of di(2-ethylhexyl) phthalate using mouse and human embryonic stem cell test models in vitro

To establish a mouse embryonic stem cell test (mEST) model and human embryonic stem cell test (hEST) model, to evaluate the embryotoxicity of di(2-ethylhexyl) phthalate (DEHP). We developed mEST and hEST models according to the European Centre for the Validation of Alternative METHODS (ECVAM). We used penicillin G (PN-G) as the standard negative reference and 5-fluorouracil (5-FU) as the standard positive reference, respectively, to verify validity of the models. Based on model validity, mouse embryonic stem cells D3 (mESC-D3), mouse Balb/c-3T3 (3T3), and human embryonic stem cells H9 (hESC-H9) were administered different concentrations of DEHP (15.6, 31.2, 62.5, 125.0, 250.0, 500.0, and 1 000.0 μg/ml) for 7 days. A cell counting Kit-8 was used to detect the 50% inhibitory proliferation concentration (IC50) of mESC-D3 cells, 3T3 cells, and hESC-H9 with DEHP. mESC-D3 and hESC-H9 were treated with DEHP (15.6, 31.2, 62.5, 125.0, 250.0 μg/ml, and 500.0 μg/ml) for 10 days based on the cytotoxicity results. At day 10, the expression of cardiomyocyte differentiation gene alpha-myosin heavy chain (α-MHC) was detected by real-time PCR and the 50% inhibition of cardiomyocycte differentiation (ID50) determined. Based on the values of IC50 and ID50, functions Ⅰ, Ⅱ and Ⅱ could be calculated by three linear discriminant functions in the EST model and the embryotoxicity of DEHP described by comparing the three functions. Nontrophoblast lineage both ES cells were cultured under optimal conditions and highly expressed hESC markers OCT4 , SSEA4, and TRA-1-60. The embryoid bodies formed were uniform in size and shape, and these results were highly repeatable. The PN-G and 5-FU results coincided with the prediction by ECVAM. Validation of our EST models was satisfactory. RESULTS of the three endpoints of DEHP in mEST were 197.3 μg/ml (IC50 3T3), 210.0 μg/ml (IC50 D3) and 246.8 μg/ml (ID50 D3). DEHP was evaluated to be a nonembryotoxic compound based on values of function Ⅰ (7.78), function Ⅱ (7.58) and function Ⅲ (-7.79). The three endpoints of DEHP in hEST were 195.4 μg/ml (IC50 3T3), 184.8 µg/ml (IC50 D3), and 84.3 µg/ml (ID50). By comparing the values of function Ⅰ (3.21), function Ⅱ (5.77), and function Ⅲ (-6.46), DEHP was evaluated to be weakly embryotoxic. DEHP was determined to be a nonembryotoxic compound by mEST and weakly embryotoxic by hEST. Therefore, hEST is a more sensible model for the evaluation of DEHP embryotoxicity.

The Extended ToxTracker Assay Discriminates Between Induction of DNA Damage, Oxidative Stress, and Protein Misfolding.

Chemical exposure of cells may damage biomolecules, cellular structures, and organelles thereby jeopardizing cellular homeostasis. A multitude of defense mechanisms have evolved that can recognize specific types of damaged molecules and will initiate distinct cellular programs aiming to remove the damage inflicted and prevent cellular havoc. As a consequence, quantitative assessment of the activity of the cellular stress responses may serve as a sensitive reporter for the induction of specific types of damage. We have previously developed the ToxTracker assay, a mammalian stem cell-based genotoxicity assay employing two green fluorescent protein reporters specific for DNA damage and oxidative stress. We have now expanded the ToxTracker assay with an additional four reporter cell lines to include monitoring of additional stress signaling pathways. This panel of six green fluorescent protein reporters is able to discriminate between different primary reactivity of chemicals being their ability to react with DNA and block DNA replication, induce oxidative stress, activate the unfolded protein response, or cause a general P53-dependent cellular stress response. Extensive validation using the compound library suggested by the European Centre for the Validation of Alternative Methods (ECVAM) and a large panel of reference chemicals shows that the ToxTracker assay has an outstanding sensitivity and specificity. In addition, we developed Toxplot, a dedicated software tool for automated data analysis and graphical representation of the test results. Rapid and reliable identification by the ToxTracker assay of specific biological reactivity can significantly improve in vitro human hazard assessment of chemicals.

Application of MatTekIn VitroReconstructed Human Skin Models for Safety, Efficacy Screening, and Basic Preclinical Research

Abstract During the past three decades, great progress has been achieved in the development of in vitro reconstructed human epidermal (RhE) tissue models. This technology is now available via various manufacturers of commercially available tissue models, and has provided opportunities for researchers worldwide to reduce or even completely avoid in vivo experimentation for screening and prediction of cutaneous toxic effects of substances and mixtures. A major step forward in recognition of RhE models as alternative tools in toxicology was their full regulatory acceptance for in vitro skin corrosion testing in 2004 as OECD Test Guideline 431. International multicenter validation studies performed during 2004–2008 under the auspices of European Centre for the Validation of Alternative Methods (ECVAM) provided further acceptance of RhE models for in vitro skin irritation testing (OECD TG 439). The use of RhE as alternatives to animal experimentation is also promoted in the new EU regulatory framework for the ...

JaCVAM-organized international validation study of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens: I. Summary of pre-validation study results

The in vivo rodent alkaline comet assay (comet assay) is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese Center for the Validation of Alternative Methods (JaCVAM), with the cooperation of the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)/the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS), organized an international validation study to evaluate the reliability and relevance of the assay for identifying genotoxic carcinogens, using liver and stomach as target organs. The ultimate goal of this validation effort was to establish an Organisation for Economic Co-operation and Development (OECD) test guideline. The purpose of the pre-validation studies (i.e., Phase 1 through 3), conducted in four or five laboratories with extensive comet assay experience, was to optimize the protocol to be used during the definitive validation study.

JaCVAM-organized international validation study of the in vivo rodent alkaline comet assay for detection of genotoxic carcinogens: II. Summary of definitive validation study results

The in vivo rodent alkaline comet assay (comet assay) is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese Center for the Validation of Alternative Methods (JaCVAM), with the cooperation of the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)/the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS), organized an international validation study to evaluate the reliability and relevance of the assay for identifying genotoxic carcinogens, using liver and stomach as target organs. The ultimate goal of this exercise was to establish an Organisation for Economic Co-operation and Development (OECD) test guideline. The study protocol was optimized in the pre-validation studies, and then the definitive (4th phase) validation study was conducted in two steps. In the 1st step, assay reproducibility was confirmed among laboratories using four coded reference chemicals and the positive control ethyl methanesulfonate. In the 2nd step, the predictive capability was investigated using 40 coded chemicals with known genotoxic and carcinogenic activity (i.e., genotoxic carcinogens, genotoxic non-carcinogens, non-genotoxic carcinogens, and non-genotoxic non-carcinogens). Based on the results obtained, the in vivo comet assay is concluded to be highly capable of identifying genotoxic chemicals and therefore can serve as a reliable predictor of rodent carcinogenicity.

Critical appraisal of alternative irritation models: three decades of testing ophthalmic pharmaceuticals.

Testing ocular tolerability of ocular pharmaceuticals is an essential regulatory requirement. The current approved reference model (gold standard) for ocular irritation testing is the Draize test. However this method is subjective and involves using live animals, hence the need to develop alternative in vitro and ex vivo testing strategies. Pubmed, Science Direct, Scopus, Google Scholar, Medline, Current Content, Web of Science and validation reports from international regulatory bodies; The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and European Centre for the Validation of Alternative Methods (ECVAM) were searched for in vitro alternatives. Whilst no single in vitro test can effectively replace the Draize eye irritation test, regulatory bodies and cosmetic/pharmaceutical industries agree that there is a need for in vitro alternatives with validated endpoints to evaluate pharmaceutical ingredients and finished eye products. There is no single in vitro test / assay that can predict the ocular irritation potential of mild to moderate test substances. This review provides a critical appraisal of the selected in vitro and ex vivo ocular toxicity models recommended by international regulatory bodies. These include cytotoxicity methods, biochemical systems and ex vivo assays. The latter are approved by ECVAM as in vitro alternatives for the well-known Draize test. Hen's egg test-chorioallantoic membrane and the isolated rabbit eye test are also accepted by regulatory agencies in France, Germany, the Netherlands and the UK. A combination of ex vivo assays along with histological examination of excised bovine cornea can predict the conjunctival and corneal tolerability and cover a wider range of ocular pharmaceutical substances.

Preliminary studies on validation of calu-3 cell line as a model for screening respiratory mucosa irritation and toxicity.

There is need to develop reproducible methods and experimental models for screening mucosal irritation and toxicity for drugs and pharmaceutical excipients. The aim of this study was to validate Calu-3 cell line as a model for screening respiratory irritation and toxicity of drugs and excipients. Eighteen test compounds were selected according to their irritation potential and European Centre for the Validation of Alternative Methods (ECVAM) guidelines. Cell toxicity and irritation was determined using MTT assay. Data analysis and interpretation were done using modified ECVAM approach; where replicate values met acceptance criteria if percent relative standard deviation (RSD) of the raw data is <18%. Compounds with mean relative viability values of 50% and below were classified as irritant (I); those above 50% were non-irritant (NI). At low concentration (0.2% w/v) and 1 h incubation, the Calu-3 cell culture model accurately predicted the toxicity of most test compounds. The specificity of our proposed model (percentage of in vivo non-irritants correctly predicted), concordance (percentage of compounds correctly predicted) and sensitivity (percentage of in vivo irritants correctly predicted) at 0.2% w/v and 60 min exposure were 100%, 72%, and 44%, respectively. In conclusion, the Calu-3 cell line in conjunction with MTT assay appears to be a potentially useful tool for screening drugs and excipients for respiratory mucosa irritation and toxicity. However, as the data reported in this study were solely based on MTT assay, additional studies are needed using other toxicity-/irritation-indicating methods to confirm the observed trend.

HSP27 as a biomarker for predicting skin irritation in human skin and reconstructed organotypic skin model

In vitro alternative tests aiming at replacing the traditional animal test for predicting the irritant potential of chemicals have been developed, but the assessing parameters or endpoints are still not sufficient. To discover novel endpoints for skin irritation responses, 2DE-based proteomics was used to analyze the protein expression in human skin exposed to sodium lauryl sulfate (SLS) following the test protocol of the European Centre for the Validation of Alternative Methods (ECVAM) in the present study. HSP27 was up-regulated most significantly among the eight identified proteins, consistent with our previous reports. Acid and basic chemicals were applied on human skin for further validation and results showed that the up-regulated expression of HSP27 was induced in 24h after the exposure. Skin-equivalent constructed with fibroblasts, basement membrane and keratinocytes was used to investigate the potential of HSP27 as a biomarker or additional endpoint for the hazard assessment of skin irritation. Our skin-equivalent (Reconstructed Organotypic Skin Model, ROSM) had excellent epidermal differentiation and was suitable for the skin irritation test. HSP27 also displayed an up-regulated expression in the ROSM in 24h after the irritants exposure for 15min. All these results suggest that HSP27 may represent a potential marker or additional endpoint for the hazard assessment of skin irritation caused by chemical products.

Performance of Comet and Micronucleus Assays in Metabolic Competent HepaRG Cells to Predict In Vivo Genotoxicity

Genetic toxicity information is critical for the safety assessment of all xenobiotics. In the absence of carcinogenicity data, genetic toxicity studies may be used to draw conclusions about the carcinogenicity potential of chemicals. However, current in vitro assays have many limitations as they produce a high rate of irrelevant positive data and possible false negative data due to the weakness of the in vitro models used. Based on the knowledge that the majority of human genotoxic carcinogens require metabolic activation to become genotoxic, it is necessary to develop in vitro cell models that mimic human liver metabolism to replace the use of liver S9 fraction, which, though helpful for predicting the potential carcinogenicity of chemicals in rodents, is questionable in humans. We therefore investigate whether the recently described human hepatoma HepaRG cells, which express the major characteristics of liver functions similarly to primary human hepatocytes, could be a suitable model for human genotoxicity assessment. We determine the performance of comet and micronucleus assays in HepaRG cells to predict in vivo genotoxins based on the list of compounds published by European Centre for the Validation of Alternative Methods (ECVAM). Twenty compounds were tested in HepaRG cells with comet and micronucleus assays over a 24-h period. The specificity, the sensitivity, and the accuracy of the two tests were determined. We found that the comet assay had higher specificity (100%) than the micronucleus (MN) test (80%), whereas the latter was far more sensitive (73%) than the former (44%), resulting nonetheless in an accuracy of 72% for the comet assay and 75% for the MN test. Taken together, our data suggest that the HepaRG cell line can be of use in genetic toxicology and that efforts to develop competent human liver cell models should be increased.

Reevaluation of the Embryonic Stem Cell Test

In recent years there has been a heightened interest in developing alternative toxicity testing methods that enhance the current system that relies almost exclusively on whole-animal testing to include in vitro assays focused on defined pathways. The embryonic stem cell test (EST) is one alternative that has been suggested for use in developmental toxicity testing. The EST utilizes the D3 mouse embryonic stem cell line to assess sensitivity of chemicals on differentiating cardiomyocytes. Additionally, a BALB/3T3 line is used to monitor cytotoxicity and compare sensitivities between adult and embryonic cells. We have reevaluated the EST nearly 10 years after formal validation by the European Center for the Validation of Alternative Methods (ECVAM) to test the stability and reliability of the cell lines in predicting developmental toxicity. Eight compounds from the ECVAM validation including the positive control, 5-fluorouracil, and the negative control, penicillin G, were tested. All eight compounds matched the classification reported during validation, indicating comparable responses and transferability of the experimental protocol. However, an increased sensitivity of the cell lines, identified by lower ID50 and IC50 values, was observed for many of the chemicals when compared to the results from the ECVAM validation.

Validation of high‐throughput genotoxicity assay screening using γH2AX in‐cell western assay on HepG2 cells

In vitro genotoxicity tests used in regulatory toxicology studies are sensitive, but the occurrence of irrelevant positive results is high compared with carcinogenicity studies in rodents. Current in vitro genotoxicity tests are also often limited by relatively low throughput. The aim of this study was to validate an in vitro genotoxic assay in a 96-well plate format that allows the simultaneous examination of cytotoxicity and genotoxicity. The test is based on the quantification of the phosphorylation of the histone H2AX (γH2AX), which reflects a global genotoxic insult, using the In-Cell Western technique. The assay was evaluated on HepG2 cells by testing a list of 61 compounds recommended by the European Center for the Validation of Alternative Methods (ECVAM), whose genotoxic potential has already been characterized. The γH2AX assay on HepG2 cell line was highly sensitive: 75% of the genotoxic compounds gave a positive result, and specific: 90-100% of nongenotoxic compounds gave negative results. Compared with the micronucleus genotoxicity assay using the same cell line and test compounds, the γH2AX assay was more sensitive and specific. In sum, the high-throughput γH2AX assay described here can accurately detect simultaneously the genotoxic and the cytotoxic potential of compounds with different modes of mutagenic action, notably those who required metabolic activation. The use of this assay in the early discovery phase of drug development may prove to be a valuable way to assess the genotoxic potential of xenobiotics.

Validation of a human cell based high-throughput genotoxicity assay ‘Anthem’s Genotoxicity screen’ using ECVAM recommended lists of genotoxic and non-genotoxic chemicals

A novel high throughput-enabled human cell based screen, Anthem’s Genotoxicity screen, was developed to achieve higher specificity for predicting in vivo genotoxins by an in vitro method. The assay employs engineered human colon carcinoma cell line; HCT116 cells that are stably engineered with three promoter–reporter cassettes such that an increased reporter activity reflects the activation of associated signaling events in a human cell. The current study focuses on the evaluation of sensitivity and specificity of Anthem’s Genotoxicity screen using 62 compounds recommended by the European Centre for the Validation of Alternative Methods (ECVAM). The concordance of Anthem’s Genotoxicity screen with in vivo tests was 95.5% with sensitivity of 95.2% and specificity of 95.7%. Thus Anthem’s Genotoxicity screen, a high-throughput mechanism based genotox indicator test can be employed by a variety of industries for rapid screening and early detection of potential genotoxins.

Use of the Cultex® Radial Flow System as an in vitro exposure method to assess acute pulmonary toxicity of fine dusts and nanoparticles with special focus on the intra- and inter-laboratory reproducibility

Exposure of the respiratory tract to airborne particles (including metal-dusts and nano-particles) is considered as a serious health hazard. For a wide range of substances basic knowledge about the toxic properties and the underlying pathomechanisms is lacking or even completely missing. Legislation demands the toxicological characterization of all chemicals placed on the market until 2018 (REACH). As toxicological in vivo data are rare with regard to acute lung toxicity or exhibit distinct limitations (e.g. inter-species differences) and legislation claims the reduction of animal experiments in general (“3R” principle), profound in vitro models have to be established and characterized to meet these requirements. In this paper we characterize a recently introduced advanced in vitro exposure system (Cultex® RFS) showing a great similarity to the physiological in vivo exposure situation for the assessment of acute pulmonary toxicity of airborne materials.Using the Cultex® RFS, human lung epithelial cells (A549 cells) were exposed to different concentrations of airborne metal dusts (nano- and microscale particles) at the air–liquid-interface (ALI). Cell viability (WST-1 assay) as a parameter of toxicity was assessed 24h after exposure with special focus on the intra- and inter-laboratory (three independent laboratories) reproducibility.Our results show the general applicability of the Cultex® RFS with regard to the requirements of the ECVAM (European Centre for the Validation of Alternative Methods) principles on test validity underlining its robustness and stability. Intra- and inter-laboratory reproducibility can be considered as sufficient if predefined quality criteria are respected. Special attention must be paid to the pure air controls that turned out to be a critical parameter for a rational interpretation of the results.Our results are encouraging and future work is planned to improve the inter-laboratory reproducibility, to consolidate the results so far and to develop a valid prediction model.

Development of a High-Throughput Gaussia Luciferase Reporter Assay for the Activation of the GADD45a Gene by Mutagens, Promutagens, Clastogens, and Aneugens

Exposure to genotoxic carcinogens leads to increased expression of the GADD45a gene in mammalian cells. This signature of genotoxic hazard has previously been exploited in the GreenScreen HC assay, in which GADD45a expression is linked to green fluorescent protein (GFP) expression in the human TK6 lymphoblastoid cell line. This article describes the development and validation of an alternative assay (“BlueScreen HC”), in which expression is linked to Gaussia luciferase (GLuc) expression, yielding a luminescent reporter, the preferred optical output in high-throughput screening. The coelentrazine substrate of GLuc is relatively unstable, and a new buffer is reported that improves its stability. A more sensitive method is demonstrated for the measurement of cell densities in the assay, using the fluorescent cyanine dye thiazole orange. A protocol amendment also allows the assessment of pro-genotoxicity using S9 liver extracts. Compounds from the European Centre for the Validation of Alternative Methods (ECVAM) recommended list for the assessment of new or improved genotoxicity assays were evaluated with and without S9 in the new assay. The new GLuc assay was as effective as the GFP assay in producing positive results for all classes of genotoxic carcinogen and negative results for all nongenotoxins tested.