This report presents recent progress made on the basic research and the use of noncoding RNAs (ncRNAs) as biomarkers in regulatory toxicology and ecotoxicology, and as analytical and therapeutic agents, as discussed at a workshop held in Málaga, Spain, on 3 and 4 March 2016. Ten presentations provided an overview on the state-of-the art ncRNA-related research, i.e. (1) the spectrum of ncRNAs known to date; (2) their function and roles in physiology and mechanisms of action; (3) issues to be taken into consideration in linking ncRNA expression profiling to phenotypic events; (4) the role of ncRNAs during cellular adaptation to external stressors, the physiological interpretation of ncRNA expression profiles and their phenotypic anchoring for substance risk assessment; (5) a critical appraisal of the hypothesis of ‘cross-kingdom RNA effects’ especially with respect to the dietary uptake of RNAs; (6) possible correlations between changes in ncRNA expression and pathological phenotypes (‘cause or consequence’); (7) the relevance of dose levels, exposure durations, no effect levels and adverse effect levels on the interpretation of ncRNA expression profiles; (8) upcoming medical applications of ncRNA therapeutic agents; (9) an overview of ncRNA expression profiling techniques; and (10) considerations for risk assessment of ncRNAs present in agricultural products and applications. These topics were reflected in further detail in a panel discussion and in extensive plenary discussions. There was agreement that an abundance of data may be obtained from ncRNA profiling, but that these data require careful evaluation and ncRNA verification to determine the utility of specific ncRNAs in hazard identification and characterisation or the role of ncRNA expression profiles in the identification and quantification of apical effects, i.e. substance-induced observable effects identified in animal experiments.
Linking changes at the molecular level (such as ncRNA expression profiling) to toxicological outcomes is considered to offer great benefits for the safety assessment of substances. Such data may facilitate moving from regulatory decision-making substantially based on apical effects to decision-making based on an understanding of toxicological processes. Furthermore, there is the potential to use ncRNAs in combination with other molecular parameters to identify early molecular / cellular events in vitro and be able to link these with apical endpoints that may be observed in vivo. Thus, ncRNA-based technologies may assist the innovation and development of new chemical products, and reduce reliance on tests using experimental animals and the costs of developing and assessing potential adverse effects of novel molecules and the time required to market innovative substances. Clearly, these goals may only be reached in the long-term since many aspects of the roles that ncRNAs play in the maintenance of cellular homeostasis or the development of phenotypic alterations are still unknown. However, basic molecular understanding of the science in this field is developing rapidly, and ncRNA-based tools are already being applied in the areas of pharmaceuticals and agrochemicals. Since the identified goals are of outstanding relevance, they should be pursued with high priority.
On the second day of the workshop, breakout sessions served to specify research needs to advance the current understanding of the role of ncRNAs in regulatory toxicology and risk assessment in order to make a first step towards the ultimate goal of using a mode of action approach in toxicity testing. Specifically, the following research topics were identified to be forwarded to the Cefic Long-range Research Initiative (LRI) research programme as priority areas for funding:
- Conduct comprehensive literature reviews and bioinformatic reanalyses of existing data sets using a consistent methodology with the aims (1) to set up a list of candidate ncRNAs (in particular miRNAs and functionally well understood ncRNAs for which research is currently most advanced) for further assessment as relevant biomarkers in toxicity studies; and (2) to identify areas of toxicology where ncRNA profiling may provide the opportunity to overcome prevailing scientific deficiencies in obtaining information that is relevant for risk assessment. Such areas of toxicology include, but are not restricted to the analysis of non-genotoxic carcinogens and reproductive / transgenerational toxins (including in utero exposure leading to adult diseases), as well as the effects of long-term low-dose exposure and challenges in addressing mixtures in toxicology.
- Develop consensus on how to conduct ncRNA expression profiling in a toxicological context, including best scientific practices (and guidelines) to collect and curate data, to analyse data and to report the outcome of studies to support applicability of the data for regulatory decision-making. This topic should include the standardisation and validation of currently available technologies in order to improve the quantitative and qualitative comparability of ncRNA datasets obtained with different technologies and to reconcile the extreme sensitivity of profiling technologies. This project should link with a currently ongoing exercise to develop a framework for the analysis of transcriptome data.
- Conduct experimental projects to evaluate the toxicological relevance of the expression profiles of selected ncRNAs. Preferably, surplus samples from, e.g. control groups existing regulatory studies should be utilised. As necessary, such retrospectively analyses of ncRNA expression profiles may be supplemented by newly conducted (e.g. 90-day) rat oral toxicity studies. The project should aim at establishing ‘normal’ (i.e. physiological) profiles that cover the biological variability of healthy individuals (the range of intra- and inter-individual variability in the levels of ncRNA expression, incl. polymorphic differences), which is likely to be affected by a multitude of parameters, including the diet. To substantiate the relevance of key ncRNAs for cell homeostasis or pathogenesis, the experimental approaches must link measured molecular events with substance-induced pathological or apical effects in a dose-dependent manner, and they should strive to differentiate between causative changes and consequential (adaptive) responses. The data from the experimental studies should be used to integrate knowledge on ncRNAs into adverse outcome pathways. Since ncRNA-based technologies are closely related to other modern technologies, such as ‘-omics’ and epigenetics, a holistic approach should be pursued to ensure that all new insights are jointly exploited in identifying adverse outcome pathways.