Qualitative and quantitative concentration-response modelling of gene co-expression networks to unlock hepatotoxic mechanisms for next generation chemical safety assessment.

Next generation risk assessment of chemicals revolves around the use of mechanistic information without animal experimentation. In this regard, toxicogenomics has proven to be a useful tool to elucidate the underlying mechanisms of adverse effects of xenobiotics. In the present study, two widely used human in vitro hepatocyte culture systems, namely primary human hepatocytes (PHH) and human hepatoma HepaRG cells, were exposed to liver toxicants known to induce liver cholestasis, steatosis or necrosis. Benchmark concentration-response modelling was applied to transcriptomics gene co-expression networks (modules) to derive benchmark concentrations (BMCs) and to gain mechanistic insight into the hepatotoxic effects. BMCs derived by concentration-response modelling of gene co-expression modules recapitulated concentration-response modelling of individual genes. Although PHH and HepaRG cells showed overlap in deregulated genes and modules by the liver toxicants, PHH demonstrated a higher responsiveness, based on the lower BMCs of co-regulated gene modules. Such BMCs can be used as transcriptomics point of departure (tPOD) for assessing module-associated cellular (stress) pathways/processes. This approach identified clear tPODs of around maximum systemic concentration (Cmax) levels for the tested drugs, while for cosmetics ingredients the BMCs were 10-100-fold higher than the estimated plasma concentrations. This approach could serve next generation risk assessment practice to identify early responsive modules at low BMCs, that could be linked to key events in liver adverse outcome pathways. In turn, this can assist in delineating potential hazards of new test chemicals using in vitro systems and used in a risk assessment when BMCs are paired with chemical exposure assessment.

Risk assessment of chemicals has traditionally been focused on animal experiments. In contrast, next generation risk assessment uses biological information obtained from experiments in cell culture models without animals to identify potential hazards. Since the liver is the main target organ of toxicity, many liver cell (hepatocyte) models have been developed and applied for hazard assessment. In this study, two widely used human hepatocyte cell models, PHH and HepaRG, were exposed to liver toxic chemicals. Biological changes in gene expression were measured in a concentration range to identify the concentration at which a biological response was perturbed using concentration response modelling. Genes belonging to the same biological process were joined based on co-expression to derive an average concentration of this process. This animal-free approach could be applied for risk assessment when biological response concentrations were related to the expected human exposure to identify potential hazard of the test chemicals.

Toxicogenomics Data for Chemical Safety Assessment and Development of New Approach Methodologies: An Adverse Outcome Pathway-Based Approach.

Mechanistic toxicology provides a powerful approach to inform on the safety of chemicals and the development of safe-by-design compounds. Although toxicogenomics supports mechanistic evaluation of chemical exposures, its implementation into the regulatory framework is hindered by uncertainties in the analysis and interpretation of such data. The use of mechanistic evidence through the adverse outcome pathway (AOP) concept is promoted for the development of new approach methodologies (NAMs) that can reduce animal experimentation. However, to unleash the full potential of AOPs and build confidence into toxicogenomics, robust associations between AOPs and patterns of molecular alteration need to be established. Systematic curation of molecular events to AOPs will create the much-needed link between toxicogenomics and systemic mechanisms depicted by the AOPs. This, in turn, will introduce novel ways of benefitting from the AOPs, including predictive models and targeted assays, while also reducing the need for multiple testing strategies. Hence, a multi-step strategy to annotate AOPs is developed, and the resulting associations are applied to successfully highlight relevant adverse outcomes for chemical exposures with strong in vitro and in vivo convergence, supporting chemical grouping and other data-driven approaches. Finally, a panel of AOP-derived in vitro biomarkers for pulmonary fibrosis (PF) is identified and experimentally validated.

Using transcriptomics, proteomics and phosphoproteomics as new approach methodology (NAM) to define biological responses for chemical safety assessment.

Omic-based technologies are of particular interest and importance for hazard identification and health risk characterization of chemicals. Their application in the new approach methodologies (NAMs) anchored on cellular toxicity pathways is based on the premise that any apical health endpoint change must be underpinned by some alterations at the omic levels. In the present study we examined the cellular responses to two chemicals, caffeine and coumarin, by generating and integrating multi-omic data from multi-dose and multi-time point transcriptomic, proteomic and phosphoproteomic experiments. We showed that the methodology presented here was able to capture the complete chain of events from the first chemical-induced changes at the phosphoproteome level, to changes in gene expression, and lastly to changes in protein abundance, each with vastly different points of departure (PODs). In HepG2 cells we found that the metabolism of lipids and general cellular stress response to be the dominant biological processes in response to caffeine and coumarin exposure, respectively. The phosphoproteomic changes were detected early in time, at very low doses and provided a fast, adaptive cellular response to chemical exposure with 7-37-fold lower points of departure comparing to the transcriptomics. Changes in protein abundance were found much less frequently than transcriptomic changes. While challenges remain, our study provides strong and novel evidence supporting the notion that these three omic technologies can be used in an integrated manner to facilitate a more complete understanding of pathway perturbations and POD determinations for risk assessment of chemical exposures.

Human biomonitoring: assessment of exposure to chemicals and their health risks: summary for decision makers

Human biomonitoring (HBM) directly measures the concentration of chemicals pollutants or their metabolitesin human fluids and tissues. As such, HBM is a reliable instrument for the assessment of human exposure to chemicals from different sources, by different pathways and during certain periods of life.

Программы биомониторинга человека: важность для защиты здоровья человека от негативного воздействия химических веществ: техническая записка

В настоящей публикации резюмируется основная информация о БМЧ: его задачах; важности информации, полученной в результате исследований БМЧ для принятия решений по регулированию химических веществ с целью минимизации негативных последствий для здоровья человека, а также о проблемах, с которыми могут столкнуться страны при выполнении национальных программ БМЧ. В ней также подчеркивается уникальная роль БМЧ в оценке пренатального воздействия, наращивании потенциала и обеспечении готовности к чрезвычайным ситуациям, связанным с загрязнением окружающей среды. Публикация предназначена в первую очередь для специалистов общественного здравоохранения, экспертов по вопросам охраны окружающей среды и регулирования химических веществ, химиков и студентов.

Human biomonitoring programmes: importance for protecting human health from negative impacts of chemicals: technical summary

This publication summarizes key information on HBM: its objectives, the value of the information obtained through HBM surveys for making decisions on chemicals management to minimize negative health impacts, and challenges countries may face when implementing national HBM programmes. It also highlights the unique value of HBM for assessing prenatal exposure, building capacity, and preparing for emergencies related to environmental pollution. This publication is aimed at public-health and health-care professionals, environmental protection and chemicals management experts, chemists, and students.

Cell-Based Chemical Safety Assessment and Therapeutic Discovery Using Array-Based Sensors.

Synthetic chemicals are widely used in food, agriculture, and medicine, making chemical safety assessments necessary for environmental exposure. In addition, the rapid determination of chemical drug efficacy and safety is a key step in therapeutic discoveries. Cell-based screening methods are non-invasive as compared with animal studies. Cellular phenotypic changes can also provide more sensitive indicators of chemical effects than conventional cell viability. Array-based cell sensors can be engineered to maximize sensitivity to changes in cell phenotypes, lowering the threshold for detecting cellular responses under external stimuli. Overall, array-based sensing can provide a robust strategy for both cell-based chemical risk assessments and therapeutics discovery.

A framework for chemical safety assessment incorporating new approach methodologies within REACH.

The long-term investment in new approach methodologies (NAMs) within the EU and other parts of the world is beginning to result in an emerging consensus of how to use information from in silico, in vitro and targeted in vivo sources to assess the safety of chemicals. However, this methodology is being adopted very slowly for regulatory purposes. Here, we have developed a framework incorporating in silico, in vitro and in vivo methods designed to meet the requirements of REACH in which both hazard and exposure can be assessed using a tiered approach. The outputs from each tier are classification categories, safe doses, and risk assessments, and progress through the tiers depends on the output from previous tiers. We have exemplified the use of the framework with three examples. The outputs were the same or more conservative than parallel assessments based on conventional studies. The framework allows a transparent and phased introduction of NAMs in chemical safety assessment and enables science-based safety decisions which provide the same level of public health protection using fewer animals, taking less time, and using less financial and expert resource. Furthermore, it would also allow new methods to be incorporated as they develop through continuous selective evolution rather than periodic revolution.

The use of Bayesian methodology in the development and validation of a tiered assessment approach towards prediction of rat acute oral toxicity.

There exists consensus that the traditional means by which safety of chemicals is assessed-namely through reliance upon apical outcomes obtained following in vivo testing-is increasingly unfit for purpose. Whilst efforts in development of suitable alternatives continue, few have achieved levels of robustness required for regulatory acceptance. An array of "new approach methodologies" (NAM) for determining toxic effect, spanning in vitro and in silico spheres, have by now emerged. It has been suggested, intuitively, that combining data obtained from across these sources might serve to enhance overall confidence in derived judgment. This concept may be formalised in the "tiered assessment" approach, whereby evidence gathered through a sequential NAM testing strategy is exploited so to infer the properties of a compound of interest. Our intention has been to provide an illustration of how such a scheme might be developed and applied within a practical setting-adopting for this purpose the endpoint of rat acute oral lethality. Bayesian statistical inference is drawn upon to enable quantification of degree of confidence that a substance might ultimately belong to one of five LD50-associated toxicity categories. Informing this is evidence acquired both from existing in silico and in vitro resources, alongside a purposely-constructed random forest model and structural alert set. Results indicate that the combination of in silico methodologies provides moderately conservative estimations of hazard, conducive for application in safety assessment, and for which levels of certainty are defined. Accordingly, scope for potential extension of approach to further toxicological endpoints is demonstrated.

Chemical safety assessment of transformation products of landfill leachate formed during the Fenton process.

Organic chemicals identified in raw landfill leachate (LL) and their transformation products (TPs), formed during Fenton treatment, were analyzed for chemical safety following REACH guidelines. The raw LL was located in the metropolitan region of Campina Grande, in northeast Brazil. We elucidated 197 unique chemical structures, including 154 compounds that were present in raw LL and 82 compounds that were detected in the treated LL, totaling 39 persistent compounds and 43 TPs. In silico models were developed to identify and prioritize the potential level of hazard/risk these compounds pose to the environment and society. The models revealed that the Fenton process improved the biodegradability of TPs. Still, a slight increase in ecotoxicological effects was observed among the compounds in treated LL compared with those present in raw LL. No differences were observed for aryl hydrocarbon receptor (AhR) and antioxidant response element (ARE) mutagenicity. Similar behavior among both raw and treated LL samples was observed for biodegradability; Tetrahymena pyriformis, Daphnia magna, Pimephales promelas and ARE, AhR, and Ames mutagenicity. Overall, our results suggest that raw and treated LL samples have similar activity profiles for all endpoints other than biodegradability.

Inherent Safety Assessment of Industrial-Scale Production of Chitosan Microbeads Modified with TiO2 Nanoparticles.

In this study, the inherent safety analysis of large-scale production of chitosan microbeads modified with TiO2 nanoparticles was developed using the Inherent Safety Index (ISI) methodology. This topology was structured based on two main stages: (i) Green-based synthesis of TiO2 nanoparticles based on lemongrass oil extraction and titanium isopropoxide (TTIP) hydrolysis, and (ii) Chitosan gelation and modification with nanoparticles. Stage (i) is divided into two subprocesses for accomplishing TiO2 synthesis, lemongrass oil extraction and TiO2 production. The plant was designed to produce 2033 t/year of chitosan microbeads, taking crude chitosan, lemongrass, and TTIP as the primary raw materials. The process was evaluated through the ISI methodology to identify improvement opportunity areas based on a diagnosis of process risks. This work used industrial-scale process inventory data of the analyzed production process from mass and energy balances and the process operating conditions. The ISI method comprises the Chemical Inherent Safety Index (CSI) and Process Inherent Safety Index (PSI) to assess a whole chemical process from a holistic perspective, and for this process, it reflected a global score of 28. Specifically, CSI and PSI delivered scores of 16 and 12, respectively. The analysis showed that the most significant risks are related to TTIP handling and its physical-chemical properties due to its toxicity and flammability. Insights about this process's safety performance were obtained, indicating higher risks than those from recommended standards.