Application of AOPs to assist regulatory assessment of chemical risks - Case studies, needs and recommendations.

While human regulatory risk assessment (RA) still largely relies on animal studies, new approach methodologies (NAMs) based on in vitro, in silico or non-mammalian alternative models are increasingly used to evaluate chemical hazards. Moreover, human epidemiological studies with biomarkers of effect (BoE) also play an invaluable role in identifying health effects associated with chemical exposures. To move towards the next generation risk assessment (NGRA), it is therefore crucial to establish bridges between NAMs and standard approaches, and to establish processes for increasing mechanistically-based biological plausibility in human studies. The Adverse Outcome Pathway (AOP) framework constitutes an important tool to address these needs but, despite a significant increase in knowledge and awareness, the use of AOPs in chemical RA remains limited. The objective of this paper is to address issues related to using AOPs in a regulatory context from various perspectives as it was discussed in a workshop organized within the European Union partnerships HBM4EU and PARC in spring 2022. The paper presents examples where the AOP framework has been proven useful for the human RA process, particularly in hazard prioritization and characterization, in integrated approaches to testing and assessment (IATA), and in the identification and validation of BoE in epidemiological studies. Nevertheless, several limitations were identified that hinder the optimal usability and acceptance of AOPs by the regulatory community including the lack of quantitative information on response-response relationships and of efficient ways to map chemical data (exposure and toxicity) onto AOPs. The paper summarizes suggestions, ongoing initiatives and third-party tools that may help to overcome these obstacles and thus assure better implementation of AOPs in the NGRA.

Assessment of the combined effects of chromium and benzene on the rat neuroendocrine and immune systems.

This study assessed the hypothalamic-pituitary-adrenocortical (HPA) axis and lymphoid organs (thymus, spleen, and bone marrow) of Wistar rats treated with a mixture of chromium and benzene. Animals were assessed at three time-points (45, 90 and 135 days) following oral mixture exposure. The hypothalamus-pituitary system was examined in light and electron microscopy. Lymphoid organs underwent a morphological assessment and the immunophenotype of splenocytes was characterized immunohistochemically using monoclonal antibodies. Splenocytes cytokine production of was determined by ELISA after Con-A stimulation. Combined exposure to chromium and benzene in average doses of 20 mg Cr (VI)/kg body weight/day and 0.6 ml benzene/kg body weight/day impaired the responsiveness of the central compartment of the HPA axis, as evidenced by functional activation of the secretory activity of the hypothalamus and pituitary gland, which was not followed by a sufficient extrusion of nonapeptides at the neurohypophysis and hypothalamic median eminence. Chromium and benzene exposure reduced the thymus mass, thymocytes count, and caused a number of structural and functional changes indicative of transient thymus involution. In the spleen, exposure to both chemicals resulted in lymphoreticular hyperplasia and plasma cell-macrophage transformation (also observed in lymph nodes). Apoptosis of thymocytes and lymphocytes was also observed in T-zones of the spleen. Notably, the effects were similar to those observed earlier for the single agents, under the same experimental conditions, without evidence of additivity.

Changes in Employment Situation and Macroeconomic Indicators Linked to Mental Health Following the Recession in Spain: A Multi-level Approach.

BACKGROUND: Periods of financial crisis are associated with higher psychological stress in the population and greater use of mental health services. This paper analyses the individual and contextual factors associated with mental health in the Spanish population in 2006, 2012 and 2017. METHOD: This was a cross-sectional, descriptive study at three timepoints: before (2006), during (2012) and after the recession (2017). The study population comprised individuals aged 16+ years old, polled for the National Health Survey. Dependent variable: psychiatric morbidity (PM). INDEPENDENT VARIABLES: 1) Individual socio-economic variables: (socio-demographic and psycho-social variables) and 2) contextual socio-economic variables (financial, public welfare services and labour market indicators). Multilevel logistic regression models with mixed effects were constructed to determine changes in PM in relation to the variables studied. RESULTS: Among women, the risk of PM increased when per capita health spending decreased and the percentage of temporary workers increased. The risk for men and women was lower when the employment rate decreased and the unemployment rate increased. CONCLUSIONS: It is possible that not only unemployment but also insecure employment entails a risk to mental health and that much of the employment created no longer guarantees basic levels of security it had achieved in previous decades.

Critical assessment and integration of separate lines of evidence for risk assessment of chemical mixtures.

Humans are exposed to multiple chemicals on a daily basis instead of to just a single chemical, yet the majority of existing toxicity data comes from single-chemical exposure. Multiple factors must be considered such as the route, concentration, duration, and the timing of exposure when determining toxicity to the organism. The need for adequate model systems (in vivo, in vitro, in silico and mathematical) is paramount for better understanding of chemical mixture toxicity. Currently, shortcomings plague each model system as investigators struggle to find the appropriate balance of rigor, reproducibility and appropriateness in mixture toxicity studies. Significant questions exist when comparing single-to mixture-chemical toxicity concerning additivity, synergism, potentiation, or antagonism. Dose/concentration relevance is a major consideration and should be subthreshold for better accuracy in toxicity assessment. Previous work was limited by the technology and methodology of the time, but recent advances have resulted in significant progress in the study of mixture toxicology. Novel technologies have added insight to data obtained from in vivo studies for predictive toxicity testing. These include new in vitro models: omics-related tools, organs-on-a-chip and 3D cell culture, and in silico methods. Taken together, all these modern methodologies improve the understanding of the multiple toxicity pathways associated with adverse outcomes (e.g., adverse outcome pathways), thus allowing investigators to better predict risks linked to exposure to chemical mixtures. As technology and knowledge advance, our ability to harness and integrate separate streams of evidence regarding outcomes associated with chemical mixture exposure improves. As many national and international organizations are currently stressing, studies on chemical mixture toxicity are of primary importance.

Integration of epidemiological findings with mechanistic evidence in regulatory pesticide risk assessment: EFSA experiences.

Toxicological risk assessment of plant protection products (PPP) is currently carried out with the principal input from regulatory toxicology studies following OECD test guidelines, with little input from epidemiological data. An EFSA-commissioned systematic review of pesticide epidemiological studies (Ntzani et al. in Literature review on epidemiological studies linking exposure to pesticides and health effects. EFSA supporting publication 2013:EN-497, 2013) revealed statistically significant associations, among others, between pesticide exposures, and Parkinson's disease and childhood leukemia. Thereafter, EFSA launched a project with a mandate for the plant protection products and their residues (PPR) Panel to set the ground for the use of epidemiological data in the risk assessment of pesticides, as requested by Regulation (EC) 1107/2009. The project culminated with the publication of two EFSA's scientific opinions on the potential contribution of experimental investigations and epidemiological studies in PPP risk assessment and with the scientific conference held on 20 November 2017, in Parma, Italy. The application of modern methodologies in exposure assessment, toxicology and epidemiology would improve the pesticide risk assessment process and support a mechanistic shift for the integration of these three disciplines under a novel paradigm in risk assessment. The application of the adverse outcome pathway (AOP) conceptual framework to this approach would contribute to gain insight into the biological plausibility of a hazard identified in epidemiological or experimental studies and would inform an Integrated Approach to Testing and Assessment (IATA) within a regulatory context.

Chemical exposure and infant leukaemia: development of an adverse outcome pathway (AOP) for aetiology and risk assessment research.

Infant leukaemia (<1 year old) is a rare disease of an in utero origin at an early phase of foetal development. Rearrangements of the mixed-lineage leukaemia (MLL) gene producing abnormal fusion proteins are the most frequent genetic/molecular findings in infant B cell-acute lymphoblastic leukaemia. In small epidemiological studies, mother/foetus exposures to some chemicals including pesticides have been associated with infant leukaemia; however, the strength of evidence and power of these studies are weak at best. Experimental in vitro or in vivo models do not sufficiently recapitulate the human disease and regulatory toxicology studies are unlikely to capture this kind of hazard. Here, we develop an adverse outcome pathway (AOP) based substantially on an analogous disease-secondary acute leukaemia caused by the topoisomerase II (topo II) poison etoposide-and on cellular and animal models. The hallmark of the AOP is the formation of MLL gene rearrangements via topo II poisoning, leading to fusion genes and ultimately acute leukaemia by global (epi)genetic dysregulation. The AOP condenses molecular, pathological, regulatory and clinical knowledge in a pragmatic, transparent and weight of evidence-based framework. This facilitates the interpretation and integration of epidemiological studies in the process of risk assessment by defining the biologically plausible causative mechanism(s). The AOP identified important gaps in the knowledge relevant to aetiology and risk assessment, including the specific embryonic target cell during the short and spatially restricted period of susceptibility, and the role of (epi)genetic features modifying the initiation and progression of the disease. Furthermore, the suggested AOP informs on a potential Integrated Approach to Testing and Assessment to address the risk caused by environmental chemicals in the future.

Human exposure to chemical mixtures: Challenges for the integration of toxicology with epidemiology data in risk assessment.

Little is known about the potential adverse effects from longterm exposure to complex mixtures at low doses, close to health-based reference values. Traditional chemical-specific risk assessment based on animal testing may be insufficient and the lack of toxicological studies on chemical mixtures remains a major regulatory challenge. Hence, new methodologies on cumulative risk assessment are being developed but still present major limitations. Evaluation of chemical mixture effects requires an integrated and systematic approach and close collaboration across different scientific fields, particularly toxicology, epidemiology, exposure science, risk assessment and statistics for a proper integration of data from all these disciplines. Well designed and conducted epidemiological studies can take advantage of this new paradigm and can provide insight to support the correlation between humans low-dose exposures and diseases, thus avoiding the uncertainty associated with extrapolation across species. In this regard, human epidemiology studies may play a significant role in the new vision of toxicity testing. However, this type of information has not been fully considered in risk assessment, mainly due to the inherent limitations of epidemiologic studies. An integrated approach of in vivo, in vitro and in silico data, together with systematic reviews or meta-analysis of high quality epidemiological studies will improve the robustness of risk assessment of chemical mixtures and will provide a stronger basis for regulatory decisions. The ultimate goal is that experimental and mechanistic data can lend support and biological plausibility to the human epidemiological observations.

Systematic reviews on neurodevelopmental and neurodegenerative disorders linked to pesticide exposure: Methodological features and impact on risk assessment.

BACKGROUND: Epidemiological data are not currently used in the risk assessment of chemical substances in a systematic and consistent manner. However, systematic reviews (SRs) could be useful for risk assessment as they appraise and synthesize the best epidemiological knowledge available. OBJECTIVES: To conduct a comprehensive literature search of SRs pertaining to pesticide exposure and various neurological outcomes, namely neurodevelopmental abnormalities, Parkinson's disease (PD) and Alzheimer's disease (AD), and to assess the potential contribution of SRs to the risk assessment process. SEARCH METHODS AND SELECTION CRITERIA: Search was conducted in PubMed and Web of Science databases and articles were selected if the following inclusion criteria were met: being a SR, published until April 2015 and without language restrictions. DATA COLLECTION AND ANALYSIS: For each neurological outcome, two review authors independently screened the search results for included studies. Data were extracted and summarized in two tables according to 16 criteria. Disagreements were resolved by discussion. MAIN RESULTS: The total number of studies identified in the first search was 65, 304 and 108 for neurodevelopment, PD and AD, respectively. From them, 8, 10 and 2 met the defined inclusion criteria for those outcomes, respectively. Overall, results suggest that prenatal exposure to organophosphates is associated with neurodevelopmental disturbances in preschool and school children. In contrast, postnatal exposures failed to show a clear effect across cohort studies. Regarding PD, 6 SRs reported statistically significant combined effect size estimates, with OR/RR ranging between 1.28 and 1.94. As for AD, 2 out of the 8 original articles included in the SRs found significant associations, with OR of 2.39 and 4.35, although the quality of the data was rather low. CONCLUSIONS: The critical appraisal of the SRs identified allowed for discussing the implications of SRs for risk assessment, along with the identification of gaps and limitations of current epidemiological studies that hinder their use for risk assessment. Recommendations are proposed to improve studies for this purpose. In particular, harmonized quantitative data (expressed in standardized units) would allow a better interpretation of results and would facilitate direct comparison of data across studies. Outcomes should be also harmonized for an accurate and reproducible measurement of adverse effects. Appropriate SRs and quantitative synthesis of the evidence should be performed regularly for a continuous update of the risk factors on health outcomes and to determine, if possible, dose-response curves for risk assessment.

Biomonitoring of arsenic, cadmium, lead, manganese and mercury in urine and hair of children living near mining and industrial areas.

Huelva (South West Spain) and its surrounding municipalities represent one of the most polluted estuaries in the world owing to the discharge of mining and industrial related pollutants in their proximity. A biomonitoring study was conducted to assess exposure to arsenic and some trace metals (cadmium, mercury, manganese and lead) in urine and scalp hair from a representative sample of children aged 6-9 years (n=261). This is the only study simultaneously analyzing those five metal elements in children urine and hair. The potential contribution of gender, water consumption, residence area and body mass index on urinary and hair metal concentrations was also studied. Urine levels of cadmium and total mercury in a proportion (25-50%) of our children population living near industrial/mining areas might have an impact on health, likely due to environmental exposure to metal pollution. The only significant correlation between urine and hair levels was found for mercury. Children living near agriculture areas showed increased levels of cadmium and manganese (in urine) and arsenic (in hair). In contrast, decreased urine Hg concentrations were observed in children living near mining areas. Girls exhibited significantly higher trace metal concentrations in hair than boys. The greatest urine arsenic concentrations were found in children drinking well/spring water. Although human hair can be a useful tool for biomonitoring temporal changes in metal concentrations, levels are not correlated with those found in urine except for total mercury, thus providing additional information.

A low cost fMRI-compatible tracking system using the Nintendo Wii remote.

It is sometimes necessary during functional magnetic resonance imaging (fMRI) experiments to capture different movements made by the subjects, e.g. to enable them to control an item or to analyze its kinematics. The aim of this work is to present an inexpensive hand tracking system suitable for use in a high field MRI environment. It works by introducing only one light-emitting diode (LED) in the magnet room, and by receiving its signal with a Nintendo Wii remote (the primary controller for the Nintendo Wii console) placed outside in the control room. Thus, it is possible to take high spatial and temporal resolution registers of a moving point that, in this case, is held by the hand. We tested it using a ball and racket virtual game inside a 3 Tesla MRI scanner to demonstrate the usefulness of the system. The results show the involvement of a number of areas (mainly occipital and frontal, but also parietal and temporal) when subjects are trying to stop an object that is approaching from a first person perspective, matching previous studies performed with related visuomotor tasks. The system presented here is easy to implement, easy to operate and does not produce important head movements or artifacts in the acquired images. Given its low cost and ready availability, the method described here is ideal for use in basic and clinical fMRI research to track one or more moving points that can correspond to limbs, fingers or any other object whose position needs to be known.