Integrated Testing Strategies Research Articles

Weight of evidence skin sensitization safety assessment using the bayesian net integrated testing strategy

Weight of evidence skin sensitization safety assessment using the bayesian net integrated testing strategy

Assessment of the skin sensitisation hazard of functional polysiloxanes and silanes in the SENS-IS assay

In the context of a larger testing programme that aimed at assessing the skin sensitisation potential of functional polysiloxanes and silanes, this investigation complements the available in vitro and in vivo data with data in the SENS-IS assay, a human in vitro 3D skin-based model. The SENS-IS assay allowed testing of all functional polysiloxanes and silanes without any solubility issues or limitations related to the multiconstituent nature of the commercial grade test substances. It appeared to encompass skin metabolism, a factor which we considered important for the skin sensitisation hazard assessment particularly of aminofunctionalised siloxanes and silanes. These three technical aspects posed significant challenges in the first part of the in vitro programme with the OECD-validated in vitro assays. The SENS-IS assay delivered promising results for this group of substances. On its own, it was the best performing model, as it did not pose any technical issues with the assay and it matched all in vivo outcomes. Considering its performance and avoidance of any limitations due to lack of solubility or chemical composition aspects, we concluded that the SENS-IS assay to be a suitable starting point for an integrated testing strategy for skin sensitisation for the group of functional polysiloxanes and silanes.

Human 3D Cultures as Models for Evaluating Magnetic Nanoparticle CNS Cytotoxicity after Short- and Repeated Long-Term Exposure.

Since nanoparticles (NPs) can translocate to the brain and impact the highly vulnerable central nervous system (CNS), novel in vitro tools for the assessment of NP-induced neurotoxicity are advocated. In this study, two types of CNS spheroids have been developed from human D384 astrocyte- and SH-SY5Y neuronal-like cells, and optimized in combination with standard assays (viability readout and cell morphology) to test neurotoxic effects caused by Fe3O4NPs, as NP-model, after short- (24–48 h; 1–100µg/ml) and long-term repeated exposure (30days; 0.1–25µg/ml). Short-term exposure of 3D-spheroids to Fe3O4NP induced cytotoxicity at 10 µg/mL in astrocytes and 25 µg/mL neurons. After long-term repeated dose regimen, spheroids showed concentration- and time-dependent cell mortality at 10 µg/mL for D384 and 0.5 µg/mL for SH-SY5Y, indicating a higher susceptibility of neurons than astrocytes. Both spheroid types displayed cell disaggregation after the first week of treatment at ≥0.1 µg/mL and becoming considerably evident at higher concentrations and over time. Recreating the 3D-spatial environment of the CNS allows cells to behave in vitro more closely to the in vivo situations, therefore providing a model that can be used as a stand-alone test or as a part of integrated testing strategies. These models could drive an improvement in the species-relevant predictivity of toxicity testing.

Guidance on risk assessment of the application of nanoscience and nanotechnologies in the food and feed chain: Part 1, human and animal health.

The European Food Safety Authority has produced this Guidance on human and animal health aspects (Part 1) of the risk assessment of nanoscience and nanotechnology applications in the food and feed chain. It covers the application areas within EFSA's remit, e.g. novel foods, food contact materials, food/feed additives and pesticides. The Guidance takes account of the new developments that have taken place since publication of the previous Guidance in 2011. Potential future developments are suggested in the scientific literature for nanoencapsulated delivery systems and nanocomposites in applications such as novel foods, food/feed additives, biocides, pesticides and food contact materials. Therefore, the Guidance has taken account of relevant new scientific studies that provide more insights to physicochemical properties, exposure assessment and hazard characterisation of nanomaterials. It specifically elaborates on physicochemical characterisation of nanomaterials in terms of how to establish whether a material is a nanomaterial, the key parameters that should be measured, the methods and techniques that can be used for characterisation of nanomaterials and their determination in complex matrices. It also details the aspects relating to exposure assessment and hazard identification and characterisation. In particular, nanospecific considerations relating to in vivo/in vitro toxicological studies are discussed and a tiered framework for toxicological testing is outlined. It describes in vitro degradation, toxicokinetics, genotoxicity as well as general issues relating to testing of nanomaterials. Depending on the initial tier results, studies may be needed to investigate reproductive and developmental toxicity, immunotoxicity, allergenicity, neurotoxicity, effects on gut microbiome and endocrine activity. The possible use of read‐across to fill data gaps as well as the potential use of integrated testing strategies and the knowledge of modes/mechanisms of action are also discussed. The Guidance proposes approaches to risk characterisation and uncertainty analysis, and provides recommendations for further research in this area.

Toxic and Genomic Influences of Inhaled Nanomaterials as a Basis for Predicting Adverse Outcome.

An immense variety of different types of engineered nanomaterials are currently being developed and increasingly applied to consumer products. Importantly, engineered nanomaterials may pose unexplored adverse health effects because of their small size. Particularly in occupational settings, the dustiness of certain engineered nanomaterials involves risk of inhalation and influences on lung function. These facts call for quick and cost-effective safety testing practices, such as that obtained through multiparametric high-throughput screening using cultured human lung cells. The predictive value of such in vitro-based testing depends partly on the effectiveness of coverage of the mechanisms underlying toxicity effects. The concept of adverse outcome pathways covers the array of causative effects starting from a molecular initiating event via cellular-, organ-, individual-, and population-level effects. Screening for adverse outcome pathway-related effects that drive the eventual toxic outcome provides a good basis for developing predictive testing methods and data-driven integrated testing strategies for hazard and risk assessment. Temporal and inherited genomic changes are likely to drive many adverse responses to engineered nanomaterials, such as multiwalled carbon nanotubes, of which one specific form has recently been evaluated as possibly carcinogenic. Here, we briefly describe current state-of-the-art strategies for analyzing and understanding genomic influences of engineered nanomaterial exposure, including the selected focus on lung disease, and strategies for using mechanistic knowledge to predict and prevent adverse outcome.

The transformics assay: first steps for the development of an integrated approach to investigate the malignant cell transformation in vitro.

The development of alternative methods to animal testing is a priority in the context of regulatory toxicology. Carcinogenesis is a field where the demand for alternative methods is particularly high. The standard rodent carcinogenicity bioassay requires a large use of animals, high costs, prolonged duration and shows several limitations, which can affect the comprehension of the human relevance of animal carcinogenesis. The cell transformation assay (CTA) has long been debated as a possible in vitro test to study carcinogenesis. This assay provides an easily detectable endpoint of oncotransformation, which can be used to anchor the exposure to the acquisition of the malignant phenotype. However, the current protocols do not provide information on either molecular key events supporting the carcinogenesis process, nor the mechanism of action of the test chemicals. In order to improve the use of this assay in the integrated testing strategy for carcinogenesis, we developed the transformics method, which combines the CTA and transcriptomics, to highlight the molecular steps leading to in vitro malignant transformation. We studied 3-methylcholanthrene (3-MCA), a genotoxic chemical able to induce in vitro cell transformation, at both transforming and subtransforming concentrations in BALB/c 3T3 cells and evaluated the gene modulation at critical steps of the experimental protocol. The results gave evidence for the potential key role of the immune system and the possible involvement of the aryl hydrocarbon receptor (AhR) pathway as the initial steps of the in vitro transformation process induced by 3-MCA, suggesting that the initiating events are related to non-genotoxic mechanisms.

Mechanism-informed read-across assessment of skin sensitizers based on SkinSensDB

Integrative testing strategies using adverse outcome pathway (AOP)-based alternative assays for assessing skin sensitizers show the potential for replacing animal testing. However, the application of alternative assays for a large number of chemicals is still time-consuming and expensive. In order to facilitate the assessment of skin sensitizers based on integrative testing strategies, a mechanism-informed read-across assessment method was proposed and evaluated using data from SkinSensDB. First, the prediction performance of two integrated testing strategy models was evaluated giving the highest area under the receiver operating characteristic curve (AUC) values of 0.928 and 0.837 for predicting human and LLNA data, respectively. The proposed read-across prediction method achieves AUC values of 0.957 and 0.802 for predicting human and LLNA data, respectively, with interpretable activation statuses of AOP events. As data grows, a better prediction performance is expected. A user-friendly tool has been constructed and integrated into SkinSensDB that is publicly accessible at http://cwtung.kmu.edu.tw/skinsensdb.

Development of an artificial neural network model for risk assessment of skin sensitization using human cell line activation test, direct peptide reactivity assay, KeratinoSens™ and in silico structure alert parameter.

It is important to predict the potential of cosmetic ingredients to cause skin sensitization, and in accordance with the European Union cosmetic directive for the replacement of animal tests, several in vitro tests based on the adverse outcome pathway have been developed for hazard identification, such as the direct peptide reactivity assay, KeratinoSens™ and the human cell line activation test. Here, we describe the development of an artificial neural network (ANN) prediction model for skin sensitization risk assessment based on the integrated testing strategy concept, using direct peptide reactivity assay, KeratinoSens™, human cell line activation test and an in silico or structure alert parameter. We first investigated the relationship between published murine local lymph node assay EC3 values, which represent skin sensitization potency, and in vitro test results using a panel of about 134 chemicals for which all the required data were available. Predictions based on ANN analysis using combinations of parameters from all three in vitro tests showed a good correlation with local lymph node assay EC3 values. However, when the ANN model was applied to a testing set of 28 chemicals that had not been included in the training set, predicted EC3s were overestimated for some chemicals. Incorporation of an additional in silico or structure alert descriptor (obtained with TIMES-M or Toxtree software) in the ANN model improved the results. Our findings suggest that the ANN model based on the integrated testing strategy concept could be useful for evaluating the skin sensitization potential.

A guide to nanosafety testing: Considerations on cytotoxicity testing in different cell models

Safety testing of nanoparticles (NPs) relies on robust, predictive, and reproducible methods. Integrated test strategies usually start with the assessment of in vitro cytotoxicity. Methods for this purpose are only partially established for NPs. The aim of this study was to evaluate three cytotoxicity assays for their applicability to NPs in several human cell models representing the most important NP target organs and to compare cell responses.The assays covered different biological principles, i.e. ATP content, redox metabolism, and membrane permeability, as well as different read-out principles, i.e. luminescence, colorimetry, and fluorescence. For all assays standard operating procedures were developed. A549 and NCI-H441 lung epithelial cells in single and in co-culture with THP-1 derived macrophages served as lung epithelial models. Normal human epidermal keratinocytes and in vitro reconstituted human epidermis were employed as skin models. Undifferentiated and differentiated Caco-2 gut epithelial cells were applied as gut epithelial models. Primary CD34-derived dendritic cells (CD34-DCs) served as an immune model. We used well-characterized model NPs, i.e. 50nm amine-modified and 50nm carboxyl-modified polystyrene NPs (PS-NH2 and PS-COOH, respectively).PS-COOH NPs showed no effect in any test while PS-NH2 displayed cytotoxicity in most cell models. CD34-DCs was the most sensitive cell model tested. The tetrazolium-based MTS assay was the most robust assay in our study, applicable to all cell models investigated. Therefore, this assay could become an integral part of a NP testing strategy. Other assays might be also useful, depending on the cell model or the type of investigation.

Applicability of an Integrated Testing Strategy consisting of in silico, in chemico and in vitro assays for evaluating the skin sensitization potencies of isocyanates

The skin sensitization potential of chemicals has been traditionally assessed using regulatory accepted in vivo methods, such as guinea pig maximization test or mouse local lymph node assays (LLNAs). A huge effort to reduce and replace the use of animals for safety assessments of chemicals because of regulatory requirements and ethical issues is presently underway, and alternative non-animal methods have been greatly developed. So far, a few studies have investigated the sensitization potencies of isocyanates which is a group of highly reactive chemicals that are known to be occupational allergens. The present study evaluated nine commonly used isocyanates using an in vivo LLNA and assessed the applicability of an Integrated Testing Strategy (ITS) consisting of an in silico Derek Nexus prediction, an in chemico direct peptide reactivity assay (DPRA), and an in vitro human Cell Line Activation Test (h-CLAT) to isocyanates. All nine isocyanates were evaluated as positive using the LLNA, Derek Nexus and DPRA, whereas seven chemicals tested positive using the h-CLAT: hexamethylene diisocyanate tested negative, and 1,5-diisocyanatonaphthalene could not be examined because of a solubility issue. When assessed using the ITS, the positive/negative evaluations of skin sensitization hazard were consistent with those assessed using the LLNA for all nine chemicals. However, the potency prediction results of the ITS tended to be underestimated, compared with those of the LLNA. The data presented in this work provide insights into the performance of non-animal testing approaches for evaluating the skin sensitization potencies of isocyanates.

Online Integrated Testing Strategy (ITS) for quantitative skin sensitization potency assessment using Bayesian networks and accounting for bioavailability

Online Integrated Testing Strategy (ITS) for quantitative skin sensitization potency assessment using Bayesian networks and accounting for bioavailability

Refinement of in silico cytogenicity evaluation and development of an integrated testing strategy for carcinogenicity based on data science

Refinement of in silico cytogenicity evaluation and development of an integrated testing strategy for carcinogenicity based on data science

Skin Sensitization: Challenging the Conventional Thinking-A Case Against 2 Out of 3 as Integrated Testing Strategy.

Skin Sensitization: Challenging the Conventional Thinking-A Case Against 2 Out of 3 as Integrated Testing Strategy.

Perspectives on In Vitro to In Vivo Extrapolations.

Quantitative in vitro to in vivo extrapolation (QIVIVE) is broadly considered a prerequisite bridge from in vitro findings to a dose paradigm. Quality and relevance of cell systems are the first prerequisite for QIVIVE. Information-rich and mechanistic endpoints (biomarkers) improve extrapolations, but a sophisticated endpoint does not make a bad cell model a good one. The next need is reverse toxicokinetics (TK), which estimates the dose necessary to reach a tissue concentration that is active in vitro. The Johns Hopkins Center for Alternatives to Animal Testing (CAAT) has created a roadmap for animal-free systemic toxicity testing, in which the needs and opportunities for TK are elaborated, in the context of different systemic toxicities. The report was discussed at two stakeholder forums in Brussels in 2012 and in Washington in 2013; the key recommendations are summarized herein. Contrary to common belief and the Paracelsus paradigm of everything is toxic, the majority of industrial chemicals do not exhibit toxicity. Strengthening the credibility of negative results of alternative approaches for hazard identification, therefore, avoids the need for QIVIVE. Here, especially the combination of methods in integrated testing strategies is most promising. Two further but very different approaches aim to overcome the problem of modeling in vivo complexity: The human-on-a-chip movement aims to reproduce large parts of living organism's complexity via microphysiological systems, that is, organ equivalents combined by microfluidics. At the same time, the Toxicity Testing in the 21st Century (Tox-21c) movement aims for mechanistic approaches (adverse outcome pathways as promoted by Organisation for Economic Co-operation and Development (OECD) or pathways of toxicity in the Human Toxome Project) for high-throughput screening, biological phenotyping, and ultimately a systems toxicology approach through integration with computer modeling. These 21st century approaches also require 21st century validation, for example, by evidence-based toxicology. Ultimately, QIVIVE is a prerequisite for extrapolating Tox-21c such approaches to human risk assessment.

Binary test battery with KeratinoSens™ and h-CLAT as part of a bottom-up approach for skin sensitization hazard prediction

Skin sensitization is one of the key safety endpoints for chemicals applied directly to the skin. Several integrated testing strategies (ITS) using multiple non-animal test methods have been developed to accurately evaluate the sensitizing potential of chemicals, but there is no regulatory-accepted ITS to classify a chemical as a non-sensitizer. In this study, the predictive performance of a binary test battery with KeratinoSens™ and h-CLAT compared to the local lymph node assay (LLNA) and human data was examined using comprehensive dataset of 203 chemicals. When two negative results indicate a non-sensitizer, the binary test battery provided sensitivity of 93.4% or 94.4% compared with the LLNA or human data. Taking into account the predictive limitations (i.e. high log Kow, pre-/pro-haptens and acyl transfer agents (or amine-reactive)), the binary test battery had extremely high sensitivity comparable to that of the 3 out of 3 ITS where three negative results of the DPRA, KeratinoSens™ and h-CLAT indicate a non-sensitizer. Therefore, the data from KeratinoSens™ or h-CLAT may provide partly redundant information on the molecular initiating event derived from DPRA. Taken together, the binary test battery of KeratinoSens™ and h-CLAT could be used as part of a bottom-up approach for skin sensitization hazard prediction.

Non‐animal assessment of skin sensitization hazard: Is an integrated testing strategy needed, and if so what should be integrated?

There is an expectation that to meet regulatory requirements, and avoid or minimize animal testing, integrated approaches to testing and assessment will be needed that rely on assays representing key events (KEs) in the skin sensitization adverse outcome pathway. Three non-animal assays have been formally validated and regulatory adopted: the direct peptide reactivity assay (DPRA), the KeratinoSens™ assay and the human cell line activation test (h-CLAT). There have been many efforts to develop integrated approaches to testing and assessment with the "two out of three" approach attracting much attention. Here a set of 271 chemicals with mouse, human and non-animal sensitization test data was evaluated to compare the predictive performances of the three individual non-animal assays, their binary combinations and the "two out of three" approach in predicting skin sensitization potential. The most predictive approach was to use both the DPRA and h-CLAT as follows: (1) perform DPRA - if positive, classify as sensitizing, and (2) if negative, perform h-CLAT - a positive outcome denotes a sensitizer, a negative, a non-sensitizer. With this approach, 85% (local lymph node assay) and 93% (human) of non-sensitizer predictions were correct, whereas the "two out of three" approach had 69% (local lymph node assay) and 79% (human) of non-sensitizer predictions correct. The findings are consistent with the argument, supported by published quantitative mechanistic models that only the first KE needs to be modeled. All three assays model this KE to an extent. The value of using more than one assay depends on how the different assays compensate for each other's technical limitations. Copyright © 2017 John Wiley & Sons, Ltd.

Integrated testing strategies can be optimal for chemical risk classification

There is an urgent need to refine strategies for testing the safety of chemical compounds. This need arises both from the financial and ethical costs of animal tests, but also from the opportunities presented by new in-vitro and in-silico alternatives. Here we explore the mathematical theory underpinning the formulation of optimal testing strategies in toxicology. We show how the costs and imprecisions of the various tests, and the variability in exposures and responses of individuals, can be assembled rationally to form a Markov Decision Problem. We compute the corresponding optimal policies using well developed theory based on Dynamic Programming, thereby identifying and overcoming some methodological and logical inconsistencies which may exist in the current toxicological testing. By illustrating our methods for two simple but readily generalisable examples we show how so-called integrated testing strategies, where information of different precisions from different sources is combined and where different initial test outcomes lead to different sets of future tests, can arise naturally as optimal policies.

Comparison and validation of an in vitro skin sensitization strategy using a data set of 33 chemical references

Allergic contact dermatitis (ACD) is an adverse health effect that develops following repeated exposure to skin sensitizing chemicals. An animal testing ban has been applied in EU, leading to development of reliably predictive non-animal methods. Several in vitro methods have been developed as alternatives but one single non-animal test method is not been sufficient to fully address since the LLNA test ban.Here, we have selected an ITS (Integrated Testing Strategy) for skin sensitization which focuses on three in vitro methods that covered the first three steps of the AOP (DPRA, SENS-IS or h-CLAT). The aim of this study was to compare these three methods due to the WoE approach based on a 2-out-of-3-assessment. The results of 33 references were compared to in vivo data (especially human). We have shown that tested firstly DPRA and SENS-IS have permitted to conclude on 29 of 33 chemicals, whereas DPRA and h-CLAT on 25, and SENS-IS and h-CLAT on 23. With this sequence, DPRA and SENS-IS and then h-CLAT in case of equivocal results, we conclude more quickly by performing fewer tests. Thereby, we have shown that it is better to follow a preferential sequence than testing chemicals simultaneously with these three methods.

Behaviour of chemical respiratory allergens in novel predictive methods for skin sensitisation

Asthma resulting from sensitisation of the respiratory tract to chemicals is an important occupational health issue, presenting many toxicological challenges. Most importantly there are no recognised predictive methods for respiratory allergens. Nevertheless, it has been found that all known chemical respiratory allergens elicit positive responses in assays for skin sensitising chemicals. Thus, chemicals failing to induce a positive response in skin sensitisation assays such as the local lymph node assay (LLNA) lack not only skin sensitising activity, but also the potential to cause respiratory sensitisation. However, it is unclear whether it will be possible to regard chemicals that are negative in in vitro skin sensitisation tests also as lacking respiratory sensitising activity. To address this, the behaviour of chemical respiratory allergens in the LLNA and in recently validated non-animal tests for skin sensitisation have been examined. Most chemical respiratory allergens are positive in one or more newly validated non-animal test methods, although the situation varies between individual assays. The use of an integrated testing strategy could provide a basis for recognition of most respiratory sensitising chemicals. However, a more complete picture of the performance characteristics of such tests is required before specific recommendations can be made.

Development of an Adverse Outcome Pathway for the Onset of Hypertension by Oxidative Stress-Mediated Perturbation of Endothelial Nitric Oxide Bioavailability

Abstract Hypertension is a cardiovascular risk factor that has a profound influence on cardiovascular morbidity and mortality. While good progress has been made in terms of identifying and managing this risk factor for patient care, methods to assess the potential of chemical compounds to induce hypertension or to assess the efficacy of consumer products (e.g., e-cigarettes) targeted at reducing disease burden remain largely limited to epidemiological associations and in vivo studies. The field of toxicology has undergone a paradigm shift toward the replacement of in vivo testing in toxicological risk assessment. The adverse outcome pathway (AOP) framework could facilitate improved knowledge-based risk/benefit assessment of chemicals or consumer products, respectively, without the necessity of animal testing. Furthermore, to facilitate a more timely, cost effective, and ethical solution for risk/efficacy assessment purposes, integrated testing strategies are required, which do not heavily rely on in vivo ...