Value and limitation of structure-based profilers to characterize developmental and reproductive toxicity potential.

The uncertainty regarding the safety of chemicals leaching from food packaging triggers attention. In silico models provide solutions for screening of these chemicals, since many are toxicologically uncharacterized. For hazard assessment, information on developmental and reproductive toxicity (DART) is needed. The possibility to apply in silico toxicology to identify and quantify DART alerts was investigated. Open-source models and profilers were applied to 195 packaging chemicals and analogues. An approach based on DART and estrogen receptor (ER) binding profilers and molecular docking was able to identify all except for one chemical with documented DART properties. Twenty percent of the chemicals in the database known to be negative in experimental studies were classified as positive. The scheme was then applied to 121 untested chemicals. Alerts were identified for sixteen of them, five being packaging substances, the others structural analogues. Read-across was then developed to translate alerts into quantitative toxicological values. They can be used to calculate margins of exposure (MoE), the size of which reflects safety concern. The application of this approach appears valuable for hazard characterization of toxicologically untested packaging migrants. It is an alternative to the use of default uncertainty factor (UF) applied to animal chronic toxicity value to handle absence of DART data in hazard characterization.

QSAR Modeling of ToxCast Assays Relevant to the Molecular Initiating Events of AOPs Leading to Hepatic Steatosis.

Nonalcoholic hepatic steatosis is a worldwide epidemiological concern since it is among the most prominent hepatic diseases. Indeed, research in toxicology and epidemiology has gathered evidence that exposure to endocrine disruptors can perturb cellular homeostasis and cause this disease. Therefore, assessing the likelihood of a chemical to trigger hepatic steatosis is a matter of the utmost importance. However, systematic in vivo testing of all the chemicals humans are exposed to is not feasible for ethical and economical reasons. In this context, predicting the molecular initiating events (MIE) leading to hepatic steatosis by QSAR modeling is an issue of practical relevance in modern toxicology. In this article, we present QSAR models based on random forest classifiers and DRAGON molecular descriptors for the prediction of in vitro assays that are relevant to MIEs leading to hepatic steatosis. These assays were provided by the ToxCast program and proved to be predictive for the detection of chemical-induced steatosis. During the modeling process, special attention was paid to chemical and toxicological data curation. We adopted two modeling strategies (undersampling and balanced random forests) to develop robust QSAR models from unbalanced data sets. The two modeling approaches gave similar results in terms of predictivity, and most of the models satisfy a minimum percentage of correctly predicted chemicals equal to 75%. Finally, and most importantly, the developed models proved to be useful as an effective in silico screening test for hepatic steatosis.

Integrating computational methods to predict mutagenicity of aromatic azo compounds.

Azo dyes have several industrial uses. However, these azo dyes and their degradation products showed mutagenicity, inducing damage in environmental and human systems. Computational methods are proposed as cheap and rapid alternatives to predict the toxicity of azo dyes. A benchmark dataset of Ames data for 354 azo dyes was employed to develop three classification strategies using knowledge-based methods and docking simulations. Results were compared and integrated with three models from the literature, developing a series of consensus strategies. The good results confirm the usefulness of in silico methods as a support for experimental methods to predict the mutagenicity of azo compounds.

Comparing the CORAL and Random Forest approaches for modelling the in vitro cytotoxicity of silica nanomaterials.

Nanotechnology is one of the most important technological developments of the 21st century. In silico methods to predict toxicity, such as quantitative structure-activity relationships (QSARs), promote the safe-by-design approach for the development of new materials, including nanomaterials. In this study, a set of cytotoxicity experimental data corresponding to 19 data points for silica nanomaterials were investigated, to compare the widely employed CORAL and Random Forest approaches in terms of their usefulness for developing so-called 'nano-QSAR' models. 'External' leave-one-out cross-validation (LOO) analysis was performed, to validate the two different approaches. An analysis of variable importance measures and signed feature contributions for both algorithms was undertaken, in order to interpret the models developed. CORAL showed a more pronounced difference between the average coefficient of determination (R²) for training and for LOO (0.83 and 0.65 for training and LOO, respectively), compared to Random Forest (0.87 and 0.78 without bootstrap sampling, 0.90 and 0.78 with bootstrap sampling), which may be due to overfitting. With regard to the physicochemical properties of the nanomaterials, the aspect ratio and zeta potential were found to be the two most important variables for Random Forest, and the average feature contributions calculated for the corresponding descriptors were consistent with the clear trends observed in the data set: less negative zeta potential values and lower aspect ratio values were associated with higher cytotoxicity. In contrast, CORAL failed to capture these trends.

Synthesis, biological evaluation, and docking studies of PAR2-AP-derived pseudopeptides as inhibitors of kallikrein 5 and 6.

A series of protease activated receptor 2 activating peptide (PAR2-AP) derivatives (1-15) were designed and synthesized. The obtained compounds were tested on a panel of human kallikreins (hKLK1, hKLK2, hKLK5, hKLK6, and hKLK7) and were found completely inactive toward hKLK1, hKLK2, and hKLK7. Aiming to investigate the mode of interaction between the most interesting compounds and the selected hKLKs, docking studies were performed. The described compounds distinguish the different human tissue kallikreins with compounds 1 and 5 as the best hKLK5 and hKLK6 inhibitors, respectively.

Hierarchical Rules for Read-Across and In Silico Models of Mutagenicity.

A broad set of rules has been implemented within the ToxRead software for read-across of chemicals for bacterial mutagenicity. These rules were obtained by manually analyzing more than 6000 chemicals and the associated chemical classes. A hierarchy of rules was established to identify those most specifically relating to the target compounds, linked in sequence to the other, more generic ones, which may match with the target compound. Rules related to both mutagenicity and lack of mutagenicity were found. Some of the latter are exceptions to the mutagenicity rules, while others are modulators of activity. These rules can also be used to predict mutagenicity, offering good performance.

Identification of a pepducin acting as S1P3 receptor antagonist.

Sphingosine-1-phosphate (S1P) is a bioactive lipid with key functions in the immune, inflammatory, and cardiovascular systems. S1P exerts its action through the interaction with a family of five known G protein-coupled receptors, named S1P(1-5). Among them, S1P(3) has been implicated in the pathological processes of a number of diseases, including sepsis and cancer. KRX-725 (compound 1) is a pepducin that mimics the effects of S1P by triggering specifically S1P(3). Here, aiming to identify novel S1P(3) antagonists, we carried out an alanine scanning analysis to address the contribution of the side chains of each amino acid residue to the peptide function. Then, deleted peptides from both the C- and N-terminus were prepared in order to determine the minimal sequence for activity and to identify the structural requirements for agonistic and, possibly, antagonistic behaviors. The pharmacological results of the Ala-scan derived compounds (2-10) suggested a high tolerance of the pepducin 1 to amino acid substitutions. Importantly, the deleted peptide 16 has the ability to inhibit, in a dose-dependent manner, both pepducin 1-induced vasorelaxation and fibroblast proliferation. Finally, a computational analysis was performed on the prepared compounds, showing that the supposed antagonists 16 and 17 appeared to be aligned with each other but not with the others. These results suggested a correlation between specific conformations and activities.

In silico approaches in carcinogenicity hazard assessment: case study of pregabalin, a nongenotoxic mouse carcinogen.

In silico toxicology protocols are meant to support computationally-based assessments using principles that ensure that results can be generated, recorded, communicated, archived, and then evaluated in a uniform, consistent, and reproducible manner. We investigated the availability of in silico models to predict the carcinogenic potential of pregabalin using the ten key characteristics of carcinogens as a framework for organizing mechanistic studies. Pregabalin is a single-species carcinogen producing only one type of tumor, hemangiosarcomas in mice via a nongenotoxic mechanism. The overall goal of this exercise is to test the ability of in silico models to predict nongenotoxic carcinogenicity with pregabalin as a case study. The established mode of action (MOA) of pregabalin is triggered by tissue hypoxia, leading to oxidative stress (KC5), chronic inflammation (KC6), and increased cell proliferation (KC10) of endothelial cells. Of these KCs, in silico models are available only for selected endpoints in KC5, limiting the usefulness of computational tools in prediction of pregabalin carcinogenicity. KC1 (electrophilicity), KC2 (genotoxicity), and KC8 (receptor-mediated effects), for which predictive in silico models exist, do not play a role in this mode of action. Confidence in the overall assessments is considered to be medium to high for KCs 1, 2, 5, 6, 7 (immune system effects), 8, and 10 (cell proliferation), largely due to the high-quality experimental data. In order to move away from dependence on animal data, development of reliable in silico models for prediction of oxidative stress, chronic inflammation, immunosuppression, and cell proliferation will be critical for the ability to predict nongenotoxic compound carcinogenicity.

Using VEGAHUB Within a Weight-of-Evidence Strategy.

Industrial needs and regulatory requirements have played a significant role in accelerating the use of nontesting methods including in silico tools as alternatives to animal testing. The main interest is not solely on the use of in silico tools, or in read-across, but on better toxicological safety assessment of substances, and for this purpose more advanced, integrated strategies have to be implemented. VEGAHUB wants to promote this broader view, not necessarily focused on a specific approach. Applying multiple tools and complementary approaches instead of one technique may provide more elements for a more robust evaluation, but at the same time it is important to have a conceptual scheme to integrate multiple, heterogeneous lines of evidence. We will show how the user can benefit from the diversity of tools available within the platform VEGAHUB for assessing the biological properties of chemical substances on an example of (non)mutagenicity.

Prediction of the Partition Coefficient between Adipose Tissue and Blood for Environmental Chemicals: From Single QSAR Models to an Integrated Approach.

The adipose tissue:blood partition coefficient is a key-endpoint to predict the pharmacokinetics of chemicals in humans and animals, since other organ:blood affinities can be estimated as a function of this parameter. We performed a search in the literature to select all the available rat in vivo data. This approach resulted into two improvements to existing models: a homogeneous definition of the endpoint and an expanded data collection. The resulting dataset was used to develop QSAR models as a function of linear and non-linear algorithms. Several applicability domain definitions were assessed and the definition corresponding to a good balance between performance and coverage was retained. We assessed the pertinence of combining single models into integrated approaches to increase the accuracy in predictions. The best integrated model outperformed the single models and it was characterized by an external mean absolute error (MAE) equal to 0.26, while preserving an adequate coverage (84 %). This performance is comparable to experimental variability and it highlights the pertinence of the integrated model.

SpheraCosmolife: a new tool for the risk assessment of cosmetic products.

A new, freely available software for cosmetic products has been designed that considers the regulatory framework for cosmetics. The software allows an overall toxicological evaluation of cosmetic ingredients without the need for additional testing and, depending on the product type, it applies defined exposure scenarios to derive risk for consumers. It takes regulatory thresholds into account and uses either experimental values, if available, or predictions. Based on the exper­imental or predicted no observed adverse effect level (NOAEL), the software can define a point of departure (POD), which is used to calculate the margin of safety (MoS) of the query chemicals. The software also provides other toxico­logical properties, such as mutagenicity, skin sensitization, and the threshold of toxicological concern (TTC) to provide an overall evaluation of the potential chemical hazard. Predictions are calculated using in silico models implemented within the VEGA software. The full list of ingredients of a cosmetic product can be processed at the same time, at the effective concentrations in the product as given by the user. SpheraCosmolife is designed as a support tool for safety assessors of cosmetic products and can be used to prioritize the cosmetic ingredients or formulations according to their potential risk to consumers. The major novelty of the tool is that it wraps a series of models (some of them new) into a single, user-friendly software system.

In Silico Approaches In Carcinogenicity Hazard Assessment: Current Status and Future Needs.

Historically, identifying carcinogens has relied primarily on tumor studies in rodents, which require enormous resources in both money and time. In silico models have been developed for predicting rodent carcinogens but have not yet found general regulatory acceptance, in part due to the lack of a generally accepted protocol for performing such an assessment as well as limitations in predictive performance and scope. There remains a need for additional, improved in silico carcinogenicity models, especially ones that are more human-relevant, for use in research and regulatory decision-making. As part of an international effort to develop in silico toxicological protocols, a consortium of toxicologists, computational scientists, and regulatory scientists across several industries and governmental agencies evaluated the extent to which in silico models exist for each of the recently defined 10 key characteristics (KCs) of carcinogens. This position paper summarizes the current status of in silico tools for the assessment of each KC and identifies the data gaps that need to be addressed before a comprehensive in silico carcinogenicity protocol can be developed for regulatory use.

CoMPARA: Collaborative Modeling Project for Androgen Receptor Activity.

BACKGROUND: Endocrine disrupting chemicals (EDCs) are xenobiotics that mimic the interaction of natural hormones and alter synthesis, transport, or metabolic pathways. The prospect of EDCs causing adverse health effects in humans and wildlife has led to the development of scientific and regulatory approaches for evaluating bioactivity. This need is being addressed using high-throughput screening (HTS) in vitro approaches and computational modeling. OBJECTIVES: In support of the Endocrine Disruptor Screening Program, the U.S. Environmental Protection Agency (EPA) led two worldwide consortiums to virtually screen chemicals for their potential estrogenic and androgenic activities. Here, we describe the Collaborative Modeling Project for Androgen Receptor Activity (CoMPARA) efforts, which follows the steps of the Collaborative Estrogen Receptor Activity Prediction Project (CERAPP). METHODS: The CoMPARA list of screened chemicals built on CERAPP's list of 32,464 chemicals to include additional chemicals of interest, as well as simulated ToxCast™ metabolites, totaling 55,450 chemical structures. Computational toxicology scientists from 25 international groups contributed 91 predictive models for binding, agonist, and antagonist activity predictions. Models were underpinned by a common training set of 1,746 chemicals compiled from a combined data set of 11 ToxCast™/Tox21 HTS in vitro assays. RESULTS: The resulting models were evaluated using curated literature data extracted from different sources. To overcome the limitations of single-model approaches, CoMPARA predictions were combined into consensus models that provided averaged predictive accuracy of approximately 80% for the evaluation set. DISCUSSION: The strengths and limitations of the consensus predictions were discussed with example chemicals; then, the models were implemented into the free and open-source OPERA application to enable screening of new chemicals with a defined applicability domain and accuracy assessment. This implementation was used to screen the entire EPA DSSTox database of ∼875,000 chemicals, and their predicted AR activities have been made available on the EPA CompTox Chemicals dashboard and National Toxicology Program's Integrated Chemical Environment. https://doi.org/10.1289/EHP5580.

Development, validation and integration of in silico models to identify androgen active chemicals.

Humans are exposed to large numbers of environmental chemicals, some of which potentially interfere with the endocrine system. The identification of potential endocrine disrupting chemicals (EDCs) has gained increasing priority in the assessment of environmental hazards. The U.S. Environmental Protection Agency (U.S. EPA) has developed the Endocrine Disruptor Screening Program (EDSP) which aims to prioritize and screen potential EDCs. The Toxicity Forecaster (ToxCast) program has generated data using in vitro high-throughput screening (HTS) assays measuring activity of chemicals at multiple points along the androgen receptor (AR) activity pathway. In the present study, using a large and diverse data set of 1667 chemicals provided by the U.S. EPA from the combined ToxCast AR assays in the framework of the Collaborative Modeling Project for Androgen Receptor Activity (CoMPARA). Two models were built using ADMET Predictor™; one is based on Artificial Neural Networks (ANNs) technology and the other uses a Support Vector Machine (SVM) algorithm; one model is a Decision Tree (DT) developed in R; and two models make use of differently combined sets of structural alerts (SAs) automatically extracted by SARpy. We used two strategies to integrate predictions from single models; one is based on a majority vote approach and the other on prediction convergence. These strategies led to enhanced statistical performance in most cases. Moreover, the majority vote approach improved prediction coverage when one or more single models were not able to provide any estimations. This study integrates multiple in silico approaches as a virtual screening tool for use in risk assessment of endocrine disrupting chemicals.

Integrated strategy for mutagenicity prediction applied to food contact chemicals.

Food contamination due to unintentional leakage of chemicals from food contact materials (FCM) is a source of increasing concern. Since for many of these substances, only limited or no toxicological data are available, the development of alternative methodologies to establish rapidly and cost-efficiently level of safety concern is critical to ensure adequate consumer protection. Computational toxicology methods are considered the most promising solutions to cope with this data gap. In particular, mutagenicity assessment has a particular relevance and is a mandatory requirement for all substances released from plastic FCM, regardless how low migration and exposure are. In the present work, a strategy integrating a number of (Quantitative) Structure Activity Relationship ((Q)SAR) models for Ames mutagenicity predictions is proposed. A list of chemicals representing likely migrating moieties from FCM was selected to test the value of the newly defined strategy and the possibility to combine predictions given by the different algorithms was evaluated. In particular, a scheme to integrate mutagenicity estimations into a single final assessment was developed resulting in an increased domain of applicability. In most cases, a deeper analysis of experimental data, where available, allowed fixing misclassification errors, highlighting the importance of data curation in the development, validation and application of in silico methods. The high accuracy of the strategy provided the rationales for its application for toxicologically uncharacterized chemicals. Finally, the overall strategy of integration will be automated through its implementation into a freely available software application.

Nano-QSAR Model for Predicting Cell Viability of Human Embryonic Kidney Cells.

Traditional Quantitative Structure-Activity Relationships (QSAR) models based on molecular descriptors as translators of chemical information show some drawbacks in predicting toxicity of nanomaterials due to their unique properties and to their nonhomogeneous structure.This chapter provides instructions on how to use CORAL, freely available software for building nano-QSAR models. CORAL makes use of descriptors based on "quasi-SMILES" representing physicochemical features and/or experimental conditions as an alternative to traditional SMILES encoding chemical structure to build up predictive nano-QSAR models for cytotoxicity.

(Q)SAR tools for priority setting: A case study with printed paper and board food contact material substances.

Over the last years, more stringent safety requirements for an increasing number of chemicals across many regulatory fields (e.g. industrial chemicals, pharmaceuticals, food, cosmetics, …) have triggered the need for an efficient screening strategy to prioritize the substances of highest concern. In this context, alternative methods such as in silico (i.e. computational) techniques gain more and more importance. In the current study, a new prioritization strategy for identifying potentially mutagenic substances was developed based on the combination of multiple (quantitative) structure-activity relationship ((Q)SAR) tools. Non-evaluated substances used in printed paper and board food contact materials (FCM) were selected for a case study. By applying our strategy, 106 out of the 1723 substances were assigned 'high priority' as they were predicted mutagenic by 4 different (Q)SAR models. Information provided within the models allowed to identify 53 substances for which Ames mutagenicity prediction already has in vitro Ames test results. For further prioritization, additional support could be obtained by applying local i.e. specific models, as demonstrated here for aromatic azo compounds, typically found in printed paper and board FCM. The strategy developed here can easily be applied to other groups of chemicals facing the same need for priority ranking.

Use of Read-Across Tools.

Read-across has become popular since the introduction of regulations, such as the European REACH regulation. This chapter provides instructions on how to use ToxRead, new freely available software for read-across analysis, and on how to interpret its output predictions for mutagenicity assessments.This tool offers two seminal sources: a set of rules/structural alerts, which may explain the toxicity, and a similarity tool, associated with a large database of chemicals with their properties.

[The Ormond's disease: an intriguing obstructive nephrouropathy]. / La malattia di Ormond: una intrigante nefrouropatia ostruttiva.

Idiopathic retroperitoneal fibrosis also known as Ormonds disease is a rare disorder characterized by the development of fibrotic tissue in the retroperitoneum involving the abdominal aorta and iliac arteries, ureters and the inferior vena cava. The aberrant tissue may compress ureters leading to obstructive nephrouropathy and renal failure, which are the most common clinical manifestations of this condition. The nephrologist is often consulted to make differential diagnosis for acute renal failure and obstructive uropathy. Ultrasounds may suggest the disease and the diagnosis will be confirmed by computed tomography or magnetic resonance, but biopsy is still the diagnostic gold standard. The aim of therapy is to remove the ureteral obstruction and prevent the progression and recurrence of the disease. After urine drainage by ureteral stents, medical long-term therapy is usually started whereas the open surgery is reserved as a last resort in selected patients. The pathophysiology of Ormond's disease is uncertain. For years the disease was considered reactive to local and /or systemic triggers with primarily involvement of abdominal aorta but at present is classified in the more broad spectrum of IgG4- Related- Disease, clinical pathological entity on autoimmune basis that can affect almost all of the body districts. This last concept has shed light on the understanding of the pathogenesis and opened new therapeutic perspectives with the use of biological agents. In this paper, on the basis of our paradigmatic clinical case of bilateral obstructive nephrouropathy associated with acute renal failure and examining the recent literature, we describe the clinical and therapeutic approach to Ormonds disease.

A knowledge-based expert rule system for predicting mutagenicity (Ames test) of aromatic amines and azo compounds.

Cancer is one of the main causes of death in Western countries, and a major issue for human health. Prolonged exposure to a number of chemicals was observed to be one of the primary causes of cancer in occupationally exposed persons. Thus, the development of tools for identifying hazardous chemicals and the increase of mechanistic understanding of their toxicity is a major goal for scientific research. We constructed a new knowledge-based expert system accounting the effect of different substituents for the prediction of mutagenicity (Ames test) of aromatic amines, a class of compounds of major concern because of their widespread application in industry. The herein presented model implements a series of user-defined structural rules extracted from a database of 616 primary aromatic amines, with their Ames test outcomes, aimed at identifying mutagenic and non-mutagenic chemicals. The chemical rationale behind such rules is discussed. Besides assessing the model's ability to correctly classify aromatic amines, its predictivity was further evaluated on a second database of 354 azo dyes, another class of chemicals of major concern, whose toxicity has been predicted on the basis of the toxicity of aromatic amines potentially generated from the metabolic reduction of the azo bond. Good performance in classification on both the amine (MCC, Matthews Correlation Coefficient=0.743) and the azo dye (MCC=0.584) datasets confirmed the predictive power of the model, and its suitability for use on a wide range of chemicals. Finally, the model was compared with a series of well-known mutagenicity predicting software. The good performance of our model compared with other mutagenicity models, especially in predicting azo dyes, confirmed the usefulness of this expert system as a reliable support to in vitro mutagenicity assays for screening and prioritization purposes. The model has been fully implemented as a KNIME workflow and is freely available for downstream users.