Breakout session 1: Role of ncRNAs in toxicology and risk assessment

Questions:

1: How to make use of ncRNA technology in toxicology and chemicals risk assessment?

What is relevant and not relevant? What do ncRNAs really tell us? How far do ncRNAs really add information to toxicology and RA? What role for ncRNAs in toxicology and chemicals RA in the next 5-10 years?

2: In light of what we know about ncRNAs – should we evolve and adapt current toxicology and the risk assessment of substances?

What do changes in ncRNA expression mean regarding the phenotype? What is relevant (e.g. cross-kingdom RNA effects)? What are the implications for industry/regulations? Are the changes in the expression of ncRNAs helpful in our understanding of MoAs?

Outcome breakout group ‘green’

Moderator: Helmut Greim, rapporteur: Alan Poole

Research activities should aim at collecting and merging available relevant information on ncRNAs, at best, by pooling data from different companies and institutions. For the time being, ncRNA-related research aiming at identifying biomarkers for regulatory toxicology and RA should not be restricted to promising miRNAs, even though current research is most advanced for this class of ncRNAs. Instead, it should also be open to possibly emerging evidence on the suitability of other classes of ncRNAs. The relevance of specific ncRNAs should be defined in studies that include both ncRNA expression analysis and phenotype assessments. This will serve to identify key ncRNAs that are relevant for cell homeostasis or pathogenesis. For this purpose, the range of normal variations of ncRNA expression should be identified, most importantly, by setting up historical control group databases. The variability observed in historical control datasets will provide an overview on the ranges of normal and abnormal expression levels, just as relevant ncRNAs may be identified. Further, the dose-response relationships of deviations from normality should be determined. Ideally, the knowledge on ncRNAs should be integrated into AOPs, e.g. by identifying the specific consequences of ncRNA alterations on different levels, including protein levels, enzyme compositions, and deviations from the phenotype. Finally, the standardisation and validation of ncRNA expression profiling technologies should be advanced, and guidelines on the reporting of data and results from ncRNA studies should be set up.

Outcome breakout group ‘red’

Moderator: Tim Gant, rapporteur: Madeleine Laffont

MiRNAs were identified as the currently most developed class of ncRNAs to be subjected to research projects investigating their applicability for RA. A comprehensive literature review should be conducted to determine specific miRNAs, and also lncRNA candidates. Criteria to determine the relevance of ncRNAs include pathological functionality, e.g. tumour promotion for oncogenic ncRNAs, and organ specificity. Such information should be evaluated to draw up a list of ncRNA candidates that may be suitable biomarkers. Research addressing the possible relevance of changes in the expression of selected miRNAs for toxicological assessments should strive to distinguish between physiological variations and pathophysiological alterations. To date, it is unclear which order of magnitude is relevant, i.e. which changes may lead to downstream effects on established target genes. Further, specific MoAs have not yet been discerned. These questions should be pursued both in literature reviews and in specific research projects. Research should also aim at determining relevant test systems. For the time being, ncRNA expression profiling should be integrated into established test methods, such as Good Laboratory Practice-compliant rat 90-day oral toxicity studies that include properly defined kinetics and (histo-)pathological evaluations. Thereby, ncRNA assessments will be performed in parallel with other readouts.

Relevant body compartments for ncRNA analysis need to be defined. This includes non-invasive (blood, urine, and other body fluids) and invasive assessments (classical additional target organs depending on prior non-invasive analysis serving to identify representative miRNAs for specific organs or for specific early events, e.g. in respect to tumour development). As the example of miRNAs involved in paracetamol hepatotoxicity reveals, miRNA effects may be investigated in a time-dependent manner already during early stages after substance administration when conventional parameters are not yet affected. In fact, this constitutes one of the advantages of miRNA profiling over conventional techniques. Also with respect to substance-induced carcinogenicity, different miRNA expression profiles are to be expected during pre-tumour and post-tumour investigations. Hence, changes in ncRNA expression should always be investigated in a time-dependent manner and they should always be related to normal expression levels.

Finally, ncRNA-related research should not be restricted to the investigation of carcinogenicity, but it should cover a wide range of toxicological endpoints. The pathological consequences and respective changes in ncRNA expression of long-term low-dose substance applications should also be addressed. An understanding on mechanisms of toxicity involving ncRNAs will form a basis to determine animal species-specific and tissue-specific differences in susceptibility to substance-induced effects.

Outcome breakout group ‘blue’

Moderator: Roland Buesen, rapporteur: Achim Aigner

Research on ncRNAs should serve to use the respective technologies to modify systems (plants, therapeutic agents) and to advance the applicability of ncRNAs as biomarkers for the toxicological assessment of (non-ncRNA-based) substances. With respect to the latter, ncRNA-related tools will most likely not be beneficial in isolation, but they will add to a weight-of-evidence during RA, for instance as biomarkers to predict carcinogenicity. It will be of great value for RA to identify ncRNAs that are related to the development of specific apical effects and to trace them in vitro and in vivo and across animal species and strains. Eventually, a given ncRNA’s predictivity of apical effects may be more important than its causality.

Research efforts to advance the applicability of modern technologies, including ncRNAs and ‘-omics’, for RA should strive for application-oriented validation. In identifying relevant ncRNAs, research should focus on their potential to serve as non-invasive biomarkers released into various body fluids. A targeted approach should also be applied in order to advance knowledge in areas of toxicology where current test methods are scientifically deficient.