Workshop Report 30

EXECUTIVE SUMMARY

This workshop explored the current state of the science on epigenetics and its role in reproductive toxicity. Experts from a range of scientific disciplines met over two days to share knowledge and brainstorm research needs in the field. The objectives and conclusions of the workshop were as follows:

Objective 1: Define epigenetics and understand its potential value for reproductive toxicology
Outcome:

Participants emphasised the need to clarify definitions and semantics. Four concordant definitions of epigenetics were cited:
“Heritable modifications, superimposed on DNA base sequence that regulate gene expression” (Jessica LaRocca).
“Heritable information governing a cell state unrelated to DNA sequence variability, or information that can be inherited from a parent cell that is not encoded in the DNA sequence” (John Greally).
“Chemical modifications of DNA that control expression of genes” (Daniele Fallin).
“Chemical modifications of chromatin (histone PTMs, ncRNAs) which affect gene expression and may be heritable, and play a role in reproductive toxicology” (Peter Alestrøm).
Discussions regarding the potential value for reproductive toxicology concluded that:
Our understanding of epigenetically-mediated toxicity is still underdeveloped. There is evidence to suggest that exposures to toxicants early in life (e.g. in utero or childhood, intergenerational effects) may result in epigenetic mechanisms that contribute to adverse health outcomes later in life.
The next step in furthering our understanding of the relationship between epigenetic change and adverse effects is to identify strong, reproducible, apical endpoints for use in models to investigate mechanisms of toxicity in vivo.
The ultimate goal would be to identify early predictive epigenetic markers causally linked with adverse apical endpoints that could help guide chemicals management decisions.
Objective 2: Understand the relationship between epigenetic change and adverse endpoints
Outcome:

It is not possible to distinguish or predetermine adaptive epigenetic effects from adverse epigenetic effects, especially when the link between epigenetic measurements and apical endpoints is not fully established. Too many epigenetic studies to date – including those reporting toxicant-induced transgenerational effects in mammals – have lacked causality determination; are underpowered; and have used only a single high dose level exposure.
Transgenerational effects should not be the focus of study at this time. Efforts to understand somatic effects and the potential consequences of environmentally-induced epigenetic effects within a single generation or between parent and child would be more helpful. This should include the following:
Ensure studies are reproducible and high confidence with interpretable results.
Determine which epigenetic alterations represent adverse changes, adaptive changes or are ‘biological noise’ (i.e. not alterations).
Identify and examine reproducible epigenetic endpoints within a mechanism/mode of action/adverse outcome pathway framework, to establish a robust correlation between an epigenetic change and an in vivo adverse outcome. These relationships must be examined across time and the dose-response continuum.
Epigenetic endpoint measurements could include one or more of the following: DNA methylation (including DNA hydroxymethylation), histone modifications, and miRNAs.
Model Systems: choices for whole organisms must be based on a thorough understanding of their advantages and limitations with regard to risk assessment in humans. Biological relevance to the human and mechanistic understanding to underpin regulatory utility is the primary driver. But cost, time and throughput criteria are also important. Validated in vitro model systems using well characterised cell lines should be included where appropriate because they can elucidate and validate important mechanistic understanding and address questions around causality. They can also provide epigenetic markers to complement in vivo studies.
Model compounds should be selected on the basis of a strong understanding of known phenotypic (apical endpoint) effects that are relevant to the hypothesis. The following were suggested: dexamethasone; phthalates; dioxin; oestrogen; copper; DES; and/or valproate as a control compound.

Objective 3: Develop a roadmap for the practical use of epigenetic studies in regulatory applications
Outcome:

Some participants felt the preparatory work to assess utility could be included in test guidelines now ― by collecting relevant tissues on a contingency basis for later retrospective analysis and comparison to apical endpoints. This could contribute to a better understanding of epigenetics (its potential role in human toxicology and the development of appropriate test methods); reduce animal use in the future; and make better use of those already involved in experimentation and regulatory safety assessments. It would also allow the collection of data on chemicals of concern and thus, in the longer term, enhance chemical safety. However, other participants agreed that the current understanding of toxicant-induced epigenetic change is still too limited for epigenetic endpoints to be formally incorporated into current test guidelines and that more research is required to demonstrate that examining epigenetic endpoints provides value in a regulatory context.
The following elements should be incorporated into the Roadmap:
Define the range of normality for epigenetic endpoints, particularly normality at the time of analysis and normality within the system.
Establish transparent guidelines to ensure that study designs include consistent and standardised data generation and management processes.
Consider in vitro systems, which have the potential for development as mechanistic test methods, but ensure that the in vitro system reliably models in vivo toxicity.
Establish whether epigenetic endpoints will provide added value (mechanistic understanding and insights and/or improved predictive capacity) to existing regulatory studies.

Objective 4: Develop a prioritised research agenda:
Outcome:

The following three research proposals were developed:
Develop in vivo intergenerational (not transgenerational) exposure models that will provide reproducible apical and epigenetic endpoints that can be used for correlative studies. Where appropriate, complement with validated in vitro studies to further elucidate and validate mechanistic understanding and markers.
Define epigenetic normality across different laboratories and across different species, taking into consideration confounding issues such as age.
Develop a “Centre of Enabling Resources in Data Analysis and Coordination” for data management and analysis standardisation.
Conclusion
Participants outlined a roadmap for the practical use of epigenetic investigations in the regulatory context.
The next step in furthering our understanding of the relationship between epigenetic change and adverse effects is to identify strong, reproducible, apical endpoints for use in models to investigate mechanisms of toxicity in vivo. Too many existing studies demonstrating epigenetic effects are of limited value because they are not reproducible and confidence in their findings is low (see talks by LaRocca, Greally, Gray, and Buesen).
Focus should be on investigating somatic effects, effects in single generations or between parent and child (i.e. intergenerational). There is little value in studying transgenerational effects at this time. These studies should be complemented by validated in vitro models to provide mechanistic information, elucidate questions around causality, and possibly lead to the development of epigenetic markers. Further, best practice guidance should be produced and disseminated so that epigenetic studies yield high confidence, high interpretability and high reproducibility.
The voluntary augmentation of current regulatory guideline studies could enable the better use of animal tissues for retrospective analysis of apical endpoints potentially associated with epigenetic mechanisms, however this will be difficult to put into practice as the benefit of epigenetics measurements is not yet known.
Three concrete research proposals, including possible model systems, were outlined for future action.

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