Workshop Report 32

Breakout session 2: Where are the research priorities and focus areas for industry and other funders?

Bruno Hubesch (Cefic LRI, Belgium) presented the LRI, a global research programme jointly conducted by the International Council of Chemical Associations (ICCA), the American Chemical Council (ACC), and Cefic. Within the Cefic LRI research programme, projects are funded in specific priority areas. The present workshop falls under the priority area ‘innovating chemical testing’. Here, Cefic LRI contributes to the development of toxicological methods and testing strategies that allow linking molecular data to human health and environmental impacts. In breakout session 2, the workshop participants were invited to formulate specific project ideas for Cefic LRI to advance within its research programme.

In the long term, all proposed project ideas should serve the 3Rs principle by eventually reducing the need for animal testing, and they should improve cost efficiency and the timely access to new product developments.

Project ideas from breakout group ‘green’

Moderator: Alan Poole, rapporteur: Nigel Gooderham

  1. Identification of gaps in toxicology that ncRNAs may contribute to filling

In certain areas of toxicology, the experimental approaches on how to obtain data are scientifically deficient. Such knowledge gaps include, but are not restricted to ways to assess non-genotoxic carcinogens and reproductive / transgenerational toxins, including in utero exposure leading to adult diseases, the effects of long-term low-dose exposure and problems in addressing mixtures in toxicology. Specific ncRNAs may contribute to filling such knowledge gaps. To determine their applicability as biomarkers for regulatory toxicology, the phenotypic consequences of different ncRNA expression levels, including a profile of the ‘normal’ situation and its variations in maintaining cellular homeostasis, remain to be elucidated.

A comprehensive literature review should be conducted to establish the state-of-the-art ncRNA research for promising topics, taking into account the areas of toxicology listed above. The systematic review should cover all available relevant information on ncRNAs (with relevant studies covering both ncRNA expression analysis and phenotype assessments). The literature review should form the basis for the design of subsequent experimental projects to identify toxicologically relevant ncRNAs. These experimental projects may be conducted in the form of case studies investigating, e.g. changes in the expression of specific ncRNAs taking into account dose-response relationships and temporal aspects. Such case studies should address whether the polymorphism of ncRNAs is likely to have functional consequences and whether specific epigenetic events govern other epigenetic events.

  1. Data fusion approaches

Data fusion approaches should be used to advance a meaningful and comprehensive interpretation of ncRNA expression profiles, ‘-omics’, epigenetics, and classical toxicological endpoints including (histo-)pathology. Even though the precise relationships between these data (or technologies) are not yet understood, they all merge into specific AOPs. Data fusion approaches will provide added value from existing information.

  1. Standardisation, verification and validation

Consensus should be developed on how to conduct ncRNA expression profiling in a toxicological and regulatory context; including best practice of reporting the outcome of such studies. This should include initiatives to improve standardisation of the respective technologies to form a basis for their verification and validation and to set up guidelines for reporting the outcome of ncRNA studies.

Taken together, the three projects serve to identify key ncRNAs that are relevant for cell homeostasis and/or pathogenesis and to advance a mechanistic understanding of toxicological pathways. The outcomes of the projects should be used to integrate knowledge on ncRNAs into AOPs, e.g. to identify the consequences of changes in the expression of a given ncRNA on the subsequent steps of a relevant AOP, including protein levels, enzyme compositions, and deviations from the phenotype.

Project ideas from breakout group ‘red’

Moderator: Reza Rasoulpour, rapporteur: Saskia van der Vies

  1. Literature survey

A comprehensive literature survey should review the state-of-the-art of the role that ncRNAs play in, e.g. tumour development and to identify key ncRNAs as predictive biomarkers of tumour formation. At least initially, well-established ncRNAs should be addressed with priority, which will most likely mainly include miRNAs. Nevertheless, the survey should not be limited to miRNAs, and optimal candidates will have to be defined with regard to tissue specificity and pathological functionality, i.e. especially tumour promoting properties. As an outcome of the literature survey, a list of candidate ncRNAs to choose from for the subsequent experimental project should be drawn up. This list may also include new, hitherto untested ncRNAs. Beyond the assessment of differences that will truly be relevant for toxicological assessments (physiological variations vs. pathological changes; see above), optimal testing systems have to be defined with regard to pathology / readout and time kinetics.

  1. Experimental project

A 90-day rat study should be performed to substantiate the relevance of selected ncRNAs as biomarkers for toxicity studies. For instance, it might be shown that a hepatotoxic substance does not change the ncRNA profile in other organ systems. Substances that are non-genotoxic carcinogens in different tissues should be selected as test substances. The study should be designed to allow the determination of dose-response relationships, and it should include ncRNA profiling of blood samples. Further, the choice of rat strain should be justified, just as the selection of 2-3 target organs (e.g. liver, thyroid, and kidney). All experiments should be run in parallel in multiple laboratories, and blinded samples should be used for miRNA analysis. The study should aim at linking ncRNA expression profiles to pathological findings (phenotypic alterations). Subsequent follow-up research should include a verification and validation of the ncRNA expression profiling by using unspecific chemicals, the inclusion of further organ systems, and a comparative analysis of miRNA expression profiles with ‘–omics’ and epigenetics techniques.

Project ideas from breakout group ‘blue’

Moderator: Jörg Hackermüller, rapporteur: Madeleine Laffont

  1. Literature or experimental project to investigate possible exposure-effects from exogenous ncRNAs

A literature survey or, if the available data is insufficient, an experimental project should further elucidate if ncRNAs that are known to affect many gene expression pathways may be taken up systemically upon oral exposure, and, if so, if they may exert gene expressional functions in the cells of the ingesting organism.

  1. Mining of existing data and literature survey to identify ncRNAs as potential biomarkers for RA to be progressed to a validation project

This project consists of 3 phases, i.e. (1) data mining and identification of candidate ncRNAs; (2) testing and validation with a focus on in vitro test systems; (3) increase of functional understanding of the candidate ncRNAs. Applying a targeted approach, the expression profiles of specific ncRNAs, e.g. miR-155, miR-122, or HOTAIR, should be determined and evaluated to improve an understanding of MoAs. Such work may also address areas of toxicology where current methods to understand MoAs are poor, (e.g. non-genotoxic carcinogenesis, immunotoxicity, reproductive / intergenerational toxicity, or low-dose and long-term toxicity). Normal ncRNA expression profiles and inter-individual variations in control groups should be determined and used to assist in the interpretation of substance-induced changes in ncRNA profiles. Generally, the project should take into account the results from relevant disease-related genomics projects, and data-sharing between different companies and public health departments should be encouraged. As an outcome of this project, new predictive biomarkers of toxicity and disease states should be identified.

  1. Best practices in bioinformatics for analysis of ncRNA data

This project should be linked with the ECETOC ‘-omics’ programme with separate consideration of ncRNA-specific principles for data analysis, as necessary.

  1. Workshop: Microbiome

Changes in the microbiome (i.e. the spectrum of (genes of) microorganisms present in a given organism) may adversely affect human health. Accordingly, external stressors that may change the microbiome may also elicit adverse effects. Against this background, a workshop may be convened to identify research needs that serve to elucidate how the microbiome may modify substance exposure by metabolising or chemically modifying these substances, and how the microbiome is modified by specific substances, including short ncRNAs.