Scientific Publications

Context: In vitro toxicology studies are increasingly being included as evidence in systematic reviews and chemical risk assessments. INVITES-IN, a tool for assessing the internal validity of in vitro studies, is currently under development. The first step in developing INVITES-IN involves the creation of an “item bank”, an overview of study assessment concepts that may be relevant to evaluating the internal validity of in vitro toxicology studies. The item bank and methodology for its creation presented in this manuscript are intended to be a general resource for supporting the development of appraisal tools for in vitro toxicology studies and potentially other study designs.

Methods: We derived the item bank from seven literature sources (one existing item bank created from a systematic review of assessment criteria for in vitro studies, and six purposively sampled study appraisal tools) and the transcripts of three focus groups. Assessment criteria plausibly relating to internal validity were abstracted from the literature sources and focus group transcripts, disaggregated into individual criteria, then normalised to express in the simplest achievable language the core issue in each criterion – an “item bank” of assessment concepts. The items were then mapped onto a set of bias domains. We conducted simple descriptive statistical analyses and visualisations to describe patterns in the dataset and developed recommendations for the use and development of the item bank.

Results: The item bank contains 405 items of potential relevance to evaluating the internal validity of in vitro toxicology studies.

Discussion: To our knowledge, this is the second item bank of any kind to have been created for toxicology studies, and the first to use focus groups as a data source alongside literature analysis. The large number of items contributed by focus group discussions suggests this is an efficient method for capturing internal validity issues that are not easily identifiable in the literature. We believe our item bank and methodology for its creation will be a useful resource for supporting the development of appraisal tools. Due to the broad applicability of many items in the item bank, it may be informative for study designs beyond the in vitro domain.

Bisphenol A (BPA), a synthetic chemical widely used in the production of polycarbonate plastic and epoxy resins, has been associated with a variety of adverse effects in humans including metabolic, immunological, reproductive, and neurodevelopmental effects, raising concern about its health impact. In the EU, it has been classified as toxic to reproduction and as an endocrine disruptor and was thus included in the candidate list of substances of very high concern (SVHC). On this basis, its use has 
been banned or restricted in some products. As a consequence, industries turned to bisphenol alternatives, such as bisphenol S (BPS) and bisphenol F (BPF), which are now found in various consumer products, as well as in human matrices at a global scale. However, due to their toxicity, these two bisphenols are in the process of being regulated. Other BPA alternatives, whose potential toxicity remains largely unknown due to a knowledge gap, have also started to be used in manufacturing processes. The gradual restriction of the use of BPA underscores the importance of understanding the potential risks associated with its alternatives to avoid regrettable substitutions. This review aims to summarize the current knowledge on the potential hazards related to BPA alternatives prioritized by European Regulatory Agencies based on their regulatory relevance and selected to be studied under the European Partnership for the Assessment of Risks from Chemicals (PARC): BPE, BPAP, BPP, BPZ, BPS-MAE, and TCBPA. The focus is on data related to toxicokinetic, endocrine disruption, immunotoxicity, developmental neurotoxicity, and genotoxicity/carcinogenicity, which were considered the most relevant endpoints to assess the hazard related to those substances. The goal here is to identify the data gaps in BPA alternatives toxicology and hence formulate the future directions that will be taken in the frame of the PARC project, which seeks also to enhance chemical risk assessment methodologies using new approach methodologies (NAMs).

While pesticide use is subject to strict regulatory oversight worldwide, it remains a main concern for environmental protection, including biodiversity conservation. This is partly due to the current regulatory approach that relies on separate assessments for each single pesticide, crop use, and non-target organism group at local scales. Such assessments tend to overlook the combined effects of overall pesticide usage at larger spatial scales. Integrative landscape-based approaches are emerging, enabling the consideration of agricultural management, the environmental characteristics, and the combined effects of pesticides applied in a same or in different crops within an area. These developments offer the opportunity to deliver informative risk predictions relevant for different decision contexts including their connection to larger spatial scales and to combine environmental risks of pesticides, with those from other environmental stressors. We discuss the needs, challenges, opportunities and available tools for implementing landscape-based approaches for prospective and retrospective pesticide Environmental Risk Assessments (ERA). A set of “building blocks” that emerged from the discussions have been integrated into a conceptual framework. The framework includes elements to facilitate its implementation, in particular: flexibility to address the needs of relevant users and stakeholders; means to address the inherent complexity of environmental systems; connections to make use of and integrate data derived from monitoring programs; and options for validation and approaches to facilitate future use in a regulatory context. The conceptual model can be applied to existing ERA methodologies, facilitating its comparability, and highlighting interoperability drivers at landscape level. The benefits of landscape-based pesticide ERA extend beyond regulation. Linking and validating risk predictions with relevant environmental impacts under a solid science-based approach will support the setting of protection goals and the formulation of sustainable agricultural strategies. Moreover, landscape ERA offers a communication tool on realistic pesticide impacts in a multistressors environment for stakeholders and citizens.

Based on toxicological evidence, children’s exposure to phthalates may contribute to altered neurodevelopment and abnormal regulation of brain-derived neurotrophic factor (BDNF). We analyzed data from five aligned studies of the Human Biomonitoring for Europe (HBM4EU) project. Ten phthalate metabolites and protein BDNF levels were measured in the urine samples of 1148 children aged 6–12 years from Italy (NACII-IT cohort), Slovakia (PCB-SK cohort), Hungary (InAirQ-HU cohort) and Norway (NEBII-NO). Serum BDNF was also available in 124 Slovenian children (CRP-SLO cohort). Children’s total, externalizing and internalizing behavioral problems were assessed using the Child Behavior Checklist at 7 years of age (only available in the NACII-IT cohort). Adjusted linear and negative binomial regression models were fitted, together with weighted quantile sum (WQS) regression models to assess phthalate mixture associations. Results showed that, in boys but not girls of the NACII-IT cohort, each natural-log-unit increase in mono-n-butyl phthalate (MnBP) and Mono(2-ethyl-5-oxohexyl) phthalate (MEOHP) was cross-sectionally associated with higher externalizing problems [incidence rate ratio (IRR): 1.20; 95% CI: 1.02, 1.42 and 1.26; 95% CI: 1.03, 1.55, respectively]. A suggestive mixture association with externalizing problems was also observed per each tertile mixture increase in the whole population (WQS—IRR = 1.15; 95% CI: 0.97, 1.36) and boys (IRR = 1.20; 95% CI: 0.96, 1.49). In NACII-IT, PCB-SK, InAirQ-HU and NEBII-NO cohorts together, urinary phthalate metabolites were strongly associated with higher urinary BDNF levels, with WQS regression confirming a mixture association in the whole population (percent change (PC) = 25.9%; 95% CI: 17.6, 34.7), in girls (PC = 18.6%; 95% CI: 7.92, 30.5) and mainly among boys (PC = 36.0%; 95% CI: 24.3, 48.9). Among CRP-SLO boys, each natural-log-unit increase in ∑DINCH concentration was associated with lower serum BDNF levels (PC: −8.8%; 95% CI: −16.7, −0.3). In the NACII-IT cohort, each natural-log-unit increase in urinary BDNF levels predicted worse internalizing scores among all children (IRR: 1.15; 95% CI: 1.00, 1.32). Results suggest that (1) children’s exposure to di-n-butyl phthalate (DnBP) and di(2-ethylhexyl) phthalate (DEHP) metabolites is associated with more externalizing problems in boys, (2) higher exposure to DINCH may associate with lower systemic BDNF levels in boys, (3) higher phthalate exposure is associated with higher urinary BDNF concentrations (although caution is needed since the possibility of a “urine concentration bias” that could also explain these associations in noncausal terms was identified) and (4) higher urinary BDNF concentrations may predict internalizing problems. Given this is the first study to examine the relationship between phthalate metabolite exposure and BDNF biomarkers, future studies are needed to validate the observed associations.

Harmful algal blooms and the toxins produced during these events are a human and environmental health concern worldwide. Saxitoxin and its derivatives are potent natural aquatic neurotoxins produced by certain freshwater cyanobacteria and marine algae species during these bloom events. Saxitoxins effects on human health are well studied, however its effects on aquatic biota are still largely unexplored. This work aims at evaluating the effects of a pulse acute exposure (24 h) of the model cladoceran Daphnia magna to 30 μg saxitoxin L⁻¹, which corresponds to the safety guideline established by the World Health Organization (WHO) for these toxins in recreational freshwaters. Saxitoxin effects were assessed through a comprehensive array of biochemical (antioxidant enzymes activity and lipid peroxidation), genotoxicity (alkaline comet assay), neurotoxicity (total cholinesterases activity), behavioral (swimming patterns), physiological (feeding rate and heart rate), and epigenetic (total 5-mC DNA methylation) biomarkers. Exposure resulted in decreased feeding rate, heart rate, total cholinesterases activity and catalase activity. Contrarily, other antioxidant enzymes, namely glutathione-S-transferases and selenium-dependent Glutathione peroxidase had their activity increased, together with lipid peroxidation levels. The enhancement of the antioxidant enzymes was not sufficient to prevent oxidative damage, as underpinned by lipid peroxidation enhancement. Accordingly, average DNA damage level was significantly increased in STX-exposed daphnids. Total DNA 5-mC level was significantly decreased in exposed organisms. Results showed that even a short-term exposure to saxitoxin causes significant effects on critical molecular and cellular pathways and modulates swimming patterns in D. magna individuals. This study highlights sub-lethal effects caused by saxitoxin in D. magna, suggesting that these toxins may represent a marked challenge to their thriving even at a concentration deemed safe for humans by the WHO.

Our study provides the most comprehensive dataset for high-precision radiogenic isotopes of lead (Pb) in blood for the western European population. It investigates their potential for elucidating the contribution of soil Pb to blood Pb using a human biomonitoring survey involving 81 adults and 4 children living in the urban area of Liège (Belgium). Soils in the area show moderate (median of 360 mg/kg) to high (95th percentile of 1000 mg/kg) Pb concentrations, due to former metal processing activities. Blood lead levels (BLL) measured in the study population are, on average, quantitatively consistent with a ∼ 20 % increase due to the exposure to Pb from soils, as estimated by a single-compartment biokinetic model. Consistently, its isotopic composition does not represent an endmember that fully accounts for the variability of Blood lead isotope (BLI) compositions measured in the study population. While some individuals show more thorogenic BLI ratios (relatively more enriched in ²⁰⁸Pb), which could be consistent with a greater exposure to local soils and/or by their country of birth, the BLI data mostly follow a trend roughly parallel to the European Standard Lead Pollution (ESLP) line, within the European leaded gasoline field, even two decades after the withdrawal of this source. Differences in BLI are probably associated with factors related to the presence of Pb in dwellings (pipes, paint) and drinking water distribution system, suggesting that the anthropogenic Pb in use, relevant to human exposure, may contain ore components of different origins, including the Australian Pb ore signature.

The focus on implementation of systematic review (SR) principles in chemical risk assessments (CRAs) is growing as it has the potential to advance the rigour and transparency of the CRAs. However, the SR and CRA communities use their own specific terminologies. Understanding the meaning of core SR and CRA terms and where they overlap is critical for application of SR methods and principles in CRAs. Moreover, it will increase the possibility for cross-sectorial collaboration, avoid misunderstandings, and improve communication among risk assessors, researchers, and policy makers.

We present a process for the cross-mapping of core CRA terms and core SR terms. Core terms for study appraisal, evidence synthesis and integration used in the SR and CRA communities will be included. The outcome will be an overview of how core SR terms map onto core CRA terms and vice versa, and a description of the relationship and conceptual overlap between the terms.

The cross-mapping is divided in three phases, where in the first phase the core SR and CRA terms will be identified. In the second phase, existing SR and CRA definitions will be mapped. In the third phase, descriptions of the relationship and conceptual overlap between the terms will be derived. The third phase will include weekly one-hour online meetings for SR and CRA experts.

We investigated the level of protection of reproductive and developmental toxicity offered through occupational exposure limits (OELs) and Derived No-Effect Levels for workers’ inhalation exposure (wDNELs). We compared coverage of substances that have a harmonised classification as reproductive toxicant 1 A or 1B (Repr.1 A/B), numerical values and scientific basis of 12 lists of OELs and wDNELs from REACH Registrants’ and the Committee for Risk Assessment. Across the 14 sources of OELs and wDNELs, 53 % of the Repr1A/B-substances had at least one exposure limit (counting groups of metals as one entry). Registrants’ wDNELs covered the largest share, 40 %. The numerical values could be highly variable for the same substance across the lists. How often reproductive toxicity is identified as the critical effect varies between the examined lists, both due to different assessments of the same substance and different substance coverage. Reviewing the margin of safety to reproductive toxicity cited in the documents, we found that 15 % of safety margins were lower to reproductive toxicity than the critical effect. To conclude, neither the REACH nor work environment legislation supply wDNELs or OELs for a substantial share of known reproductive toxicants. EU OELs cover among the fewest substances in the range, and in many cases national OELs or wDNELs are set at more conservative levels.