Data exploration and knowledge extraction: their application to the study of endocrine disrupting chemicals

Interest in computer-aided methods for investigating the biological field has increased significantly. One method is Quantitative Structure-Activity Relationships (QSAR), a valuable technique for predicting the effects of a substance from its chemical structure. A challenging application of QSAR is in characterizing the (bio)activity profiles of chemicals. Endocrine disrupters (EDs) are exogenous substances interfering with the function of the endocrine system and represent an interesting field of application for in silico methods. EDs targets include nuclear receptors, particularly effects mediated by the oestrogen receptor (ER). They are also mentioned as substances requiring a more detailed control and specific authorisation within REACH, the new European legislation on chemicals. QSAR represents a challenging method to approach data gap about EDs since REACH substantially boosted interest on computational chemistry to replace experimental testing. This work: aimed to explore the status, availability and reliability of non-testing methods applied to endocrine disruption via oestrogen receptors and eventually to propose new models easily exploitable in regulatory contexts. The work evaluated existing QSAR models present in literature to assess their validity on the basis of the OECD principles for QSAR validation. Different kinds of models have been analysed and they were externally validated with new data found in the literature. Furthermore, new QSAR binary classifiers have been developed using different data mining techniques (e.g.: classification trees, fuzzy logic, neural networks) based on a very large and heterogeneous dataset of chemical compounds. The focus was given to both binding (RBA) and transcriptional activity (RA) better to characterize the effects of EDs. A possible combination of the models was also explored. A very good accuracy was achieved for both RA and RBA (>85%). These models can be a valuable complement to in vivo and in vitro studies in the toxicological characterisation of chemical compounds

QSAR models for the (eco-)toxicological characterization and prioritization of emerging pollutants: case studies and potential applications within REACH.

Under the European REACH regulation (Registration, Evaluation, Authorisation and Restriction of Chemical substances - (EC) No 1907/2006), there is an urgent need to acquire a large amount of information necessary to assess and manage the potential risk of thousands of industrial chemicals. Meanwhile, REACH aims at reducing animal testing by promoting the intelligent and integrated use of alternative methods, such as in vitro testing and in silico techniques. Among these methods, models based on quantitative structure-activity relationships (QSAR) are useful tools to fill data gaps and to support the hazard and risk assessment of chemicals. The present thesis was performed in the context of the CADASTER Project (CAse studies on the Development and Application of in-Silico Techniques for Environmental hazard and Risk assessment), which aims to integrate in-silico models (e.g. QSARs) in risk assessment procedures, by showing how to increase the use of non-testing information for regulatory decision-making under REACH. The aim of this thesis was the development of QSAR/QSPR models for the characterization of the (eco-)toxicological profile and environmental behaviour of chemical substances of emerging concern. The attention was focused on four classes of compounds studied within the CADASTER project, i.e. brominated flame retardants (BFRs), fragrances, prefluorinated compounds (PFCs) and (benzo)-triazoles (B-TAZs), for which limited amount of experimental data is currently available, especially for the basic endpoints required in regulation for the hazard and risk assessment. Through several case-studies, the present thesis showed how QSAR models can be applied for the optimization of experimental testing as well as to provide useful information for the safety assessment of chemicals and support decision-making. In the first case-study, simple multiple linear regression (MLR) and classification models were developed ad hoc for BFRs and PFCs to predict specific endpoints related to endocrine disrupting (ED) potential (e.g. dioxin-like activity, estrogenic and androgenic receptor binding, interference with thyroxin transport and estradiol metabolism). The analysis of modelling molecular descriptors allowed to highlight some structural features and important structural alerts responsible for increasing specific ED activities. The developed models were applied to screen over 200 BFRs and 33 PFCs without experimental data, and to prioritize the most hazardous chemicals (on the basis of ED potency profile), which have been then suggested to other CADASTER partners in order to focus the experimental testing. In the second case-study, MLR models have been developed, specifically for B-TAZs, for the prediction of three key endpoints required in regulation to assess aquatic toxicity, i.e. acute toxicity in algae (EC50 72h Pseudokirchneriella subcapitata), daphnids (EC50 48h Daphnia magna) and fish (LC50 96h Onchorynchus mykiss). Also in this case, the developed QSARs were applied for screening purposes. Among over 350 B-TAZs lacking experimental data, 20 compounds, which were predicted as toxic (EC(LC)50 64 10 mg/L) or very toxic (EC(LC)50 64 1 mg/L) to the three aquatic species, were prioritized for further experimental testing. Finally, in the third case-study, classification QSPR models were developed for the prediction of ready biodegradability of fragrance materials. Ready biodegradation is among the basic endpoints required for the assessment of environmental persistence of chemicals. When compared with some existing models commonly used for predicting biodegradation, the here proposed QSPRs showed higher classification accuracy toward fragrance materials. This comparison highlighted the importance of using local models when dealing with specific classes of chemicals. All the proposed QSARs have been developed on the basis of the OECD principles for QSAR acceptability for regulatory purposes, paying particular attention to the external validation procedure and to the statistical definition of the applicability domain of the models. QSAR models based on molecular descriptors generated by both commercial (DRAGON) and freely-available (PaDELDescriptor, QSPR-Thesaurus) software have been proposed. The use of free tool allows for a wider applicability of the here proposed QSAR models. Concluding, the QSAR models developed within this thesis are useful tools to support hazard and risk assessment of specific classes of emerging pollutants, and show how non-testing information can be used for regulatory decisions, thus minimizing costs, time and saving animal lives. Beyond their use for regulatory purposes, the here proposed QSARs can find application in the rational design of new safer compounds that are potentially less hazardous for human health and environment

Alternative methods for regulatory toxicology – a state-of-the-art review

This state-of-the art review is based on the final report of a project carried out by the European Commission’s Joint Research Centre (JRC) for the European Chemicals Agency (ECHA). The aim of the project was to review the state of the science of non-standard methods that are available for assessing the toxicological and ecotoxicological properties of chemicals. Non-standard methods refer to alternatives to animal experiments, such as in vitro tests and computational models, as well as animal methods that are not covered by current regulatory guidelines. This report therefore reviews the current scientific status of non-standard methods for a range of human health and ecotoxicological endpoints, and provides a commentary on the mechanistic basis and regulatory applicability of these methods. For completeness, and to provide context, currently accepted (standard) methods are also summarised. In particular, the following human health endpoints are covered: a) skin irritation and corrosion; b) serious eye damage and eye irritation; c) skin sensitisation; d) acute systemic toxicity; e) repeat dose toxicity; f) genotoxicity and mutagenicity; g) carcinogenicity; h) reproductive toxicity (including effects on development and fertility); i) endocrine disruption relevant to human health; and j) toxicokinetics. In relation to ecotoxicological endpoints, the report focuses on non-standard methods for acute and chronic fish toxicity. While specific reference is made to the information needs of REACH, the Biocidal Products Regulation and the Classification, Labelling and Packaging Regulation, this review is also expected to be informative in relation to the possible use of alternative and non-standard methods in other sectors, such as cosmetics and plant protection products.JRC.I.5-Systems Toxicolog

Scientific Opinion on the hazard assessment of endocrine disruptors: Scientific criteria for identification of endocrine disruptors and appropriateness of existing test methods for assessing effects mediated by these substances on human health and the environment

Upon request of the European Commission, the Scientific Committee (SC) of the European Food Safety Authority reviewed existing information related to the testing and assessment of endocrine active substances (EASs) and endocrine disruptors (EDs). This work was conducted by a working group of experts in endocrinology, risk assessment and toxicology, together with observers from other EU agencies, namely EMA, ECHA and EEA. To distinguish between EDs and other groups of substances with different modes of action, it was concluded that an ED is defined by three criteria: the presence of i) an adverse effect in an intact organism or a (sub)population; ii) an endocrine activity; and iii) a plausible causal relationship between the two. As scientific criteria for adversity have not been generally defined, specific criteria for endocrine disrupting effects could not be identified. Hence, expert judgement is required to assess on a case-by-case basis the (eco)toxicological relevance of changes at the molecular to individual and/or (sub)population level following exposure to an EAS. The SC concluded that a reasonably complete suite of standardised assays for testing the effects of EASs is (or will soon be) available for the oestrogenic, androgenic, thyroid and steroidogenic modalities in mammals and fish, with fewer tests for birds and amphibians. Shortcomings in current tests and for other endocrine modalities and species were reviewed. Critical effect, severity, (ir)reversibility and potency aspects are part of the hazard characterisation of EDs. To inform on risk and level of concern for the purpose of risk management decisions, risk assessment (taking into account hazard and exposure data/predictions) makes best use of available information. Levels of concern are not determined exclusively by risk assessment but also by protection goals set by the risk management

Investigation of the sorption of 17α-ethynylestradiol (EE2) on soils formed under aerobic and anaerobic conditions

A study was conducted on the sorption of 17 alpha-ethynylestradiol (EE2) on five soils formed under different redox conditions: an Arenosol (A_20) with fully aerobic conditions, two Gleysol samples (G_20 and G_40) with suboxic and anoxic conditions and two Histosols (H_20 and H_80) with mostly anoxic conditions. The soils were characterized on the basis of total organic carbon (TOC), specific surface area (SSA) and the Fourier transform infrared spectra of the humic acid and humin fractions (the soil remaining after alkali extraction) of the soil. The maximum adsorption capacity of the soils (Q(max)) ranged from 10.7 to 83.6 mg/g in the order G_20 > H_20 > G_40 > A_20 > H_80, which reflected the organic matter content of the soils. The sorption isotherms were found to be nonlinear for all the soil samples, with Freundlich n values of 0.45-0.68. The strong nonlinearity found in the adsorption of the H_80 samples could be attributed to their high hard carbon content, which was confirmed by the high aromaticity of the humin fraction. The maximum sorption capacity (Q(max)) of the soils did not increase indefinitely as the organic carbon content of the soils rose. There could be two reasons for this: (i) the large amount of organic matter may reduce the number of binding sites on the surface, and (ii) the decrease in SSA with increasing soil OC content may limit the ability to adsorb EE2 molecules. In anaerobic soil samples, where organic matter accumulation is pronounced, the amount of aromatic and phenolic compounds was higher than in better aerated soil profiles. Strong correlations were found between the amount of aromatic and phenolic compounds in the organic matter and the adsorption of EE2 molecules, indicating that pi-pi interaction and H-bonding are the dominant sorption mechanisms. (C) 2019 The Authors. Published by Elsevier Ltd

Developing magnetic functionalized multi-walled carbon nanotubes-based buckypaper for the removal of Furazolid

Magnetic f-MWCNTs-based BP/PVA membrane was fabricated and utilized for the elimination of furazolidone (FZD) from aqueous solution. Characterisation and adsorption studies were performed to evaluate the performance and adsorptive efficiency, respectively of the membrane. Furthermore, statistical and machine learning technique were also applied to predict the removal efficiency of FZD on the membrane. The results revealed that magnetic f-MWCNTs-based BP/PVA membrane has the potential to be used as an efficient membrane for practical applications

The role of biosolids-derived dissolved organic matter on the interaction of selected endocrine-disrupting chemicals with two alluvial soils

Overpopulation is one of the major causes of many environmental problems we experience today. With increasing population, the demand for quality food and clean water is becoming difficult to realize. Although with advancement in technology, we are still able to cope with the demand, but eventually we will exhaust the supply for quality food and clean water as well as the different methods of achieving them. Quality food and clean water are necessities to live. To help in delivering these needs, federal regulations and policies were developed and implemented to protect the public from environmental contaminants that might be hazardous to human health. Contaminants such as organic compounds are ubiquitous in the environment because they are component of most goods that the public use on a daily basis. When these organic compounds are released into the environment they could undergo different processes such volatilization, microbial degradation, photodegradation, movement by run-off, adsorbed and held strongly in the soil, or move with the soil-water. When a hazardous organic compound is strongly held in the soil, it is possible that it will be taken-up by crops for human consumption or as feedstock. Organic compounds that are mobile in the water may also be taken-up by aquatic species, will bioaccumulate, or leach to the groundwater contaminating the source of the drinking water. A group of compounds found to disrupt the endocrine systems that brought a great concern to the scientific community was observed three decades ago. These compounds are collectively referred to as endocrine disrupting chemicals (EDC) and sometimes endocrine active compounds (EAC). Since then, various research studies have been conducted to potentially cover all aspects of EDCs. To establish the presence, and the spatial and temporal distribution of EDCs, monitoring studies of surface and even groundwater were often conducted. The effect of EDCs to wildlife, such as the feminization of frogs and fishes, and the potential health problems to humans were also intense research areas that involves EDCs. To determine the overall environmental risk of EDCs, the fate and transport of these compounds need to be evaluated as well. The fate and transport studies will explore the movement of these compounds in soil, water, and air. Also, how the physical and chemical properties of these compounds changes as they interact with other chemicals, microorganisms, and other components of the soil-water systems, such as the native dissolved organic matter (soil) and the exogenous dissolved organic matter (biosolids-derived). EDCs maybe released from residential, industrial, and agricultural sources. In wastewater treatment plant, the EDCs and other contaminants may not be completely removed during the treatment process and ends up in the biosolids and effluents. To recycle the carbon and the plant nutrients such as nitrogen and phosphorus, the biosolids are applied in agricultural fields and the effluents are used as irrigation water in relatively dry regions. The fate and transport study of EDCs is not well-explored. Since there are a lot of chemicals that are manufactured and released daily, and a lot of variability in the systems condition (ex. temperature, moisture, and pH) that impact the fate and transport of the EDCs, generating more data will definitely help in establishing more reliable results. Adsorption and column transport experiments are some of the processes used to predict the fate and transport of EDCs. In this study, three endocrine disrupting chemicals (EDCs), bisphenol-A (BPA), 17α-ethinylestradiol (EE2), and 4-nonylphenol (4-NP), were chosen because of their varying physico-chemical properties. Among the three compounds, BPA is the least hydrophobic and 4-NP is the most hydrophobic. We are hoping that these compounds can be used as model compounds to study the potential environmental risk of other organic pollutants with similar physico-chemical properties that are released in soil-water system. The impact on the transport of EDCs were also investigated in systems where the native dissolved organic matter and exogenous biosolids-derived dissolved organic matter (BDOM) were present. The concentrations of BPA, EE2, and 4-NP were monitored for six months at two wastewater treatment plants in Iowa and at their corresponding upstream and downstream discharge locations. The monitoring was conducted to assess if the three EDCs of interest were actually present in the wastewater effluent and water system. Results showed that BPA, EE2, and 4-NP were detectable in the water samples, although the frequency of detection was variable throughout the six months. The result also showed that the concentrations detected were below the method detection limit (MDL), and suggested that the analytical method adopted for the monitoring studies of environmental concentrations of organic contaminants were an important consideration in conducting this type of analysis. Soil materials are complicated matrices by themselves and in combination with water and biosolids-derived dissolved organic matter, the system becomes even more complex. Also, studying the interactions and fates of the three EDCs simultaneously in soil-water-BDOM systems is challenging, and therefore simplified systems, i.e., working with individual EDCs and interaction with individual system component (BDOM-carbon to soil, EDCs to soil adsorption-desorption, EDC to BDOM adsorption) was employed in this study. The two soils used are Hanlon and Zook soil samples. The two soil samples have contrasting properties. Of the two soils, the Zook soil sample has higher total organic carbon, total organic nitrogen, and higher clay content. The biosolids-derived dissolved organic matter that was extracted from the anaerobically digested biosolids from the Ames Wastewater Pollution Control Facility was characterized and was dominated by N-acetylated carbohydrates and aromatic components. Adsorption of BDOM to the two soils revealed the predicted maximum adsorption capacity and intensity was 188 mg C kg-1 soil and 0.015 L kg-1 soil, respectively for Hanlon soil sample and 640 mg C kg-1 soil and 0.015 L kg-1 soil, respectively for the Zook soil sample. Although the Zook soil sample had a higher maximum adsorption capacity, the intensity of adsorption for both soils was low and of the same magnitude, which suggests that the BDOM-carbon is likely to be mobile in saturated conditions. Some components of the BDOM-carbon were adsorbed to the soil samples, and fractionation of the BDOM-carbon revealed that hydrophobic acid (HoA), hydrophobic neutral (HoN), and hydrophilic base (HiB) structural fractions dominated the adsorption. Some adsorption mechanisms proposed were cation bridging, hydrophobic partitioning, and electrostatic attraction. The parameters determined to predict the behavior of the EDCs adsorbed to the two soil samples were described by the Freundlich-Langmuir model. This model allowed us to calculate the n parameter or the index of heterogeneity of sorption energy. The difference between the n values limits our ability to compare sorption capacities of the two soils. For example, for EE2, the n values calculated were 0.73 and 1.93 for the Hanlon and Zook soils samples, respectively. Among the three EDCs, the 4-NP had the highest adsorption maximum capacity (Qmax) and adsorption affinity constant (KLF) predicted based on the combined Langmuir-Freundlich model for both soil samples. Between the two soil samples, the Zook soil sample had a maximum adsorption capacity about 50 times higher and an adsorption affinity about ten times higher than the Hanlon soil samples. The EE2 had a higher adsorption affinity than the BPA, but the BPA had a higher maximum adsorption capacity than EE2. The BPA exhibited about the same magnitude of adsorption capacity and adsorption affinity for both soil samples. 4-NP, the most hydrophobic EDC, had the highest adsorption capacity and affinity, while the least hydrophobic BPA had the lowest adsorption affinity. The extent of binding between the EDC and the BDOM is important because it predicts the EDC-BDOM complex formation. The formation of a complex between the EDC and BDOM helps in understanding the role of the BDOM in transport, e.g., enhancing or deterring the movement of EDCs in the soil-water system. The results showed that BPA is strongly associated with the BDOM fraction, while EE2 and 4-NP did not form a strong association with the BDOM. Considering the other adsorption results from the concurrent study, we hypothesized that the BDOM will carry with it the BPA as it moves in the soil water, while the EE2 and 4-NP will most likely interact with the soil materials. To test the hypothesis of the influence of BDOM on the transport of BPA, a packed soil column experiment was conducted using the Hanlon and Zook soil samples. There were two concentration levels of BDOM used: 10 mg C L-1 and 40 mg C L-1. The BDOM was mixed with the BPA first before passing it through the soil column. The results showed that, contrary to the hypothesis that the BDOM would enhance the transport of BPA, the presence of exogenous BDOM enhanced the retention of the BPA in both soil samples. The data generated from this research project will be a valuable source of information for future studies of organic pollutants with similar physico-chemical properties. The result could also serve as a basis to improve the federal and state policy and regulatory frameworks pertaining to the direct discharge of organic chemicals from residential and industrial areas, land application of biosolids, and the re-use of effluent as irrigation water. The monitoring study showed detection of the three EDCs but the frequency of detection varied at each month of sampling. The result of monitoring could be used as a baseline data when in the future, the focus of monitoring study is on the different seasons of sampling (temporal variability). Estrogens and BPA were detected at polluting levels in groundwater (Focazio et al., 2008; Adeel et al., 2017). One process to reclaim the groundwater is soil aquifer treatment (Mansell et al., 2003). The results generated from this study could be used to improve the efficiency of the treatment process because the removal mechanisms is most likely influenced by hydrophobic or hydrophilic characteristic of the carbon structural fraction of the adsorbent used (Mansell et al., 2003). Also, we can improve the technology in wastewater treatment processes to eliminate most of the EDCs before they are released. Part of the waste treatment process is the use of soil as component of the bedding material that adsorbs the organic contaminants. The adsorption intensity, adsorption capacity, and retardation parameter values could therefore be a used to improve the adsorptive capability of the bedding materials used

The Use of Computational Methods in the Toxicological Assessment of Chemicals in Food: Current Status and Future Prospects

A wide range of chemicals are intentionally added to, or unintentially found in, food products, often in very small amounts. Depending on the situation, the experimental data needed to complete a dietary risk assessment, which is the scientific basis for protecting human health, may not be available or obtainable, for reasons of cost, time and animal welfare. For example, toxicity data are often lacking for the metabolites and degradation products of pesticide active ingredients. There is therefore an interest in the development and application of efficient and effective non-animal methods for assessing chemical toxicity, including Quantitative Structure-Activity Relationship (QSAR) models and related computational methods. This report gives an overview of how computational methods are currently used in the field of food safety by national regulatory bodies, international advisory organisations and the food industry. On the basis of an international survey, a comprehensive literature review and a detailed QSAR analysis, a range of recommendations are made with the long-term aim of promoting the judicious use of suitable QSAR methods. The current status of QSAR methods is reviewed not only for toxicological endpoints relevant to dietary risk assessment, but also for Absorption, Distribution, Metabolism and Excretion (ADME) properties, which are often important in discriminating between the toxicological profiles of parent compounds and their reaction products. By referring to the concept of the Threshold of Toxicological Concern (TTC), the risk assessment context in which QSAR methods can be expected to be used is also discussed. This Joint Research Centre (JRC) Reference Report provides a summary and update of the findings obtained in a study carried out by the JRC under the terms of a contract awarded by the European Food Safety Authority (EFSA).JRC.DG.I.6-Systems toxicolog