Limitations of regulatory in vivo developmental neurotoxicity (DNT) testing and assessment are well known, such as the 3Rs conflict, low throughput, high costs, high specific expertise needed and the lack of deeper mechanistic information. Moreover, the standard in vivo DNT data variability and in the experimental animal to human real life extrapolation is uncertain. Here, knowledge about these limitations and uncertainties is systematically summarized using a tabular OECD format. We also outline a hypothesis how alternative, fit-for-purpose Integrated Approaches to Testing and Assessment (IATAs) for DNT could improve current standard animal testing: Relative gains in 3Rs compliance, reduced costs, higher throughput, improved basic study design, higher standardization of testing and assessment and validation without 3Rs conflict, increasing the availability and reliability of DNT data. This could allow a more reliable comparative toxicity assessment over a larger proportion of chemicals within our global environment. The use of early, mechanistic, sensitive indicators for potential DNT could better support human safety assessment and mixture extrapolation. Using kinetic modelling ideally these could provide - eventually context dependent - at least the same level of human health protection. Such new approaches could also lead to a new mechanistic understanding for chemical safety, permitting determination of a dose that is likely not to trigger defined toxicity traits or pathways, rather than a dose not causing the current apical organism endpoints. The manuscript shall motivate and guide the development of new alternative methods for IATAs with diverse applications and support decision-making for their regulatory acceptance.

With recent rapid advancement of methodological tools, mechanistic understanding of biological processes leading to carcinogenesis is expanding. New approach methodologies such as transcriptomics can inform on non-genotoxic mechanisms of chemical carcinogens and can be developed for regulatory applications. The Organisation for the Economic Cooperation and Development (OECD) expert group developing an Integrated Approach to the Testing and Assessment (IATA) of Non-Genotoxic Carcinogens (NGTxC) is reviewing the possible assays to be integrated therein. In this context, we review the application of transcriptomics approaches suitable for pre-screening gene expression changes associated with phenotypic alterations that underlie the carcinogenic processes for subsequent prioritisation of downstream test methods appropriate to specific key events of non-genotoxic carcinogenesis. Using case studies, we evaluate the potential of gene expression analyses especially in relation to breast cancer, to identify the most relevant approaches that could be utilised as (pre-) screening tools, for example Gene Set Enrichment Analysis (GSEA). We also consider how to address the challenges to integrate gene panels and transcriptomic assays into the IATA, highlighting the pivotal omics markers identified for assay measurement in the IATA key events of inflammation, immune response, mitogenic signalling and cell injury.

Information about acute fish toxicity is routinely required in many jurisdictions for environmental risk assessment of chemicals. This information is typically obtained using a 96-hour juvenile fish test for lethality according to OECD test guideline (TG) 203 or equivalent regional guidelines. However, TG 203 has never been validated using criteria currently required for new test methods including alternative methods. Characterization of the practicality and validity of TG 203 is important to provide a benchmark for alternative methods. This contribution systematically summarizes the available knowledge about limitations and uncertainties of TG 203, based on methodological, statistical, and biological considerations. Uncertainties stem from the historic flexibility (e.g. use of a broad range of species) and constraints of the basic test design (e.g., no replication). Other sources of uncertainty arise from environmental safety extrapolation based on TG 203 data. Environmental extrapolation models, combined with data from alternative methods, including mechanistic indicators of toxicity, may provide at least the same level of environmental protection. Yet, most importantly the 3R advantages of alternative methods allow a better standardization, characterization and an improved basic study design. This can enhance data reliability and thus facilitate the comparison of chemical toxicity, as well as the environmental classifications and prediction of no-effect concentrations of chemicals. Combined with the 3R gains and the potential for higher throughput, a reliable assessment of more chemicals can be achieved, leading to improved environmental protection.

Assessment of potential impacts of chemicals on the environment traditionally involves regulatory standard data requirements for acute aquatic toxicity testing using algae, daphnids and fish (e.g., OECD test guidelines (TG) 201, 202, and 203, respectively), representing different trophic levels. In line with the societal goal to replace or reduce vertebrate animal testing, alternative bioassays were developed to replace testing with fish: the fish cell line RTgill-W1 acute toxicity assay (OECD TG249) and the zebrafish embryo acute toxicity test (zFET, OECD TG236). However, previous studies revealed the lower sensitivity of the RTgill-W1 cell line assay and zFET for some neurotoxic chemicals and allyl alcohol, which is presumably biotransformed in fish to the more toxic acrolein (which is predicted well through the cell line assay). To provide an additional alternative to acute fish toxicity, in this study, we analyzed historic ecotoxicity data for fish and daphnids from the EnviroTox Database. We found a considerable variability in acute fish LC50 and acute daphnids EC50 values, particularly for neurotoxic chemicals. Comparing sensitivity of these taxonomic groups according to different neurotoxicity classification schemes indicates that fish rarely represent the most sensitive trophic level of the two. Exceptions here most prominently include a few cyclodiene compounds, which are no longer marketed, and a chemical group that could be identified through structural alerts. Moreover, daphnids are more sensitive than fish to acrolein. This analysis highlights the potential of the Daphnia acute toxicity test, which is usually a standard regulatory data requirement, in safeguarding the environmental protection level provided by the RTgill-W1 cell line assay and the zFET. This research, rooted in decades of efforts to replace the fish acute toxicity test, shifts the focus from predicting fish toxicity 1-to-1 to emphasizing the protectiveness of alternative methods, paving the way for further eliminating vertebrate tests in environmental toxicology.

Exposure to environmental chemicals has been linked to an increased risk of heart failure, but the impact on left ventricular dysfunction (LVD) markers remains uncertain. To establish a foundation of evidence regarding early HF markers and their association with environmental pollutants, a systematic review and meta-analysis was conducted. The search, conducted on October 13th, 2023, encompassed PubMed, Embase, and Web of Science without filters, focusing on observational studies reporting myocardial geometrical, structural, or functional alterations in individuals without a history of heart disease. This included the general adult population, workers, young people, and the elderly. The risk of bias was assessed using the ROBINS-I tool at both study and item levels. The systematic review included 17 studies involving 43.358 individuals exposed to air pollution and 2038 exposed to heavy metals. Approximately 41% of the effect measures of associations reported significant abnormalities in myocardial structure or function. The metanalyses by pollutants categories indicated positive associations between LV systolic and diastolic abnormalities and exposure to PM2.5 [-0.069 (-0.104, -0.033); -0.044 (-0.062, -0.025)] and PM10 [-0.055 (-0.087, -0.022); -0.030 (-0.050, -0.010)] and NO2 [-0.042 (-0.071, -0.013); -0.021 (-0.037, -0.004)], as well as positive associations between lead exposure and LV systolic abnormalities [-0.033 (-0.051, -0.016)]. To strengthen confidence in the findings and improve the attribution of baseline exposures, the authors recommend conducting more longitudinal and case-control studies. These studies should consider co-exposures, account for vulnerable groups, and identify cardiotoxic compounds that may require regulation. When examining the link between myocardial abnormalities and environmental exposure, it is also advisable to explore the supportive use of Adverse Outcome Pathway (AOP) approaches to confirm a causal relationship.