Over the past 10 years or more CEFIC LRI has supported research to better understand the health impact of hormone active agents and help develop internationally approved and validated screening and testing methods at the OECD. In November 2006 a joint CEFIC LRI & ECETOC Workshop was convened to explore opportunities for using genomic technologies to advance research on hormone active substances in particular those causing male reproductive toxicity.
A priority topic identified at the Workshop is how to apply data from genomic research into toxicological risk assessment practices in particular how toxicogenomics might be applied to recognize, and differentiate between, adverse and non-adverse effects in toxicological studies. Uncertainties about the application of toxicogenomics to develop dose response relationships applies not only to studies of male reproductive health but presents a generic challenge on how to integrate the new technologies into toxicological risk assessment. A specific challenge is agreeing how toxicogenomics should be used to accurately identify thresholds of toxicity for regulatory decisions. Currently a major component of toxicological investigations is to identify the highest exposure level, (dose or concentration), that does not cause a treatment-related toxicological effect believed to be relevant for human health. Over the years a generic, structured approach for evaluating data generated in toxicology studies has been developed using the concepts of thresholds of toxicity which differentiates between an adverse effect and adaptive change(s) in the experimental animal which although related to treatment are not considered to be adverse. A similar robust and scientifically acceptable framework is required when using data from genomic technologies to identify and differentiate between adverse and non-adverse effects.
Assuming toxicogenomic thresholds can be established it should be possible to integrate toxicogenomics with traditional quantitative outcomes of toxicity studies is shown in Figure 1.
This RfP has been developed based on the priority topics for research identified during the Workshop and includes investigations to determine:
- If toxicogenomic responses produced by exposures to hormone active agents are predictive of sub-chronic or chronic toxicological effects, and
- If there are ”toxicogenomic thresholds” which must be exceeded to progress from a normal state to an adaptive state and subsequently to an adverse /toxic state.
Responses to this RfP are therefore requested to use dose response studies which will include both standard toxicological endpoints, (such as histopathology, hormone levels), and alterations in gene response as markers to differentiate between normal, adaptive and toxic responses in the male reproductive tissues of rats following treatment with estrogenic active substances. The research will also examine the concept of toxicogenomic thresholds and determine how data from genomic research can be integrated and applied into current toxicological risk assessment practices. It is expected the data from such research will supplement developing knowledge for using genomic technologies in risk assessment being pursued by organizations such as ILSI Health & Environmental Sciences Institute (ILSI HESI). While ILSI HESI is assessing liver, kidney or gene toxic effects using micro-array technology this research proposal will focus particularly on male reproductive health providing a mechanistic based risk assessment of adverse health outcomes mediated via an endocrine mode of action.
- Demonstrate gene expression data can be evaluated in the context of traditional toxicological end-points in scientifically dependable and robust framework to determine dose response characteristics for safety assessment.
- Develop publicly available data as a resource to help shape scientific and regulatory policy on the use of toxicogenomics for toxicological risk assessment.
- Augment scientific data being developed by other organizations and engage in dialogue with researchers advancing the use of toxicogenomics in risk assessment
Short interim reports on progress are required at 3 to 6-monthly intervals. It is expected that the findings will developed into a peer reviewed publication, following presentation at a suitable scientific conference.
Research will identify changes in gene expression and toxicological effects in male reproductive tissue following exposure to estrogenic active chemicals (e.g. 17ß-estradiol) in experimental rats. The investigation will build on current knowledge on gene expression in male reproductive tissues produced by estrogenic agents and toxicological effects associated with exposure to such agents. It is anticipated the genes chosen will be specific to the male reproductive tract and/or relevant to the mode of action and toxicological effects of the substances investigated. In addition, data derived from such investigations must be shown to be reproducible.
For convenience the research can be viewed as being in three parts:
- The first part will establish background variations in normal gene expression including effects of experimental variables such as age, diet and strain.
- The second part will focus on time- and dose-dependent progressions marking thresholds of toxicity from ”no effect” to ”adaptive change” to ”adverse/toxicological effect” to determine if progression from normal, through adaptive to adverse measured by traditional toxicological endpoints (e.g. histopathology and changes in hormone levels) is reflected in toxicogenomic thresholds which must be exceeded for the cascade of change to progress.
- The third part will be analysis of data for biomarkers of endocrine toxicity relevant to male reproductive health by reference to the toxicological endpoint data and including any other relevant genomic data which may be available in the public domain at the time of analysis.
The proposal should include examination of well characterized male reproductive toxicants which mediate toxic effects via estrogenic and non-estrogenic modes of action. Dose response curves need to be established for each chemical investigated and should include one dose where there is a clear adverse effect as measured by traditional approaches (e.g. histopathology, enzyme induction, reproductive toxicity etc.). Negative controls should also be included.
While it is expected the first part of the study (investigating normal variation in gene expression) will include a broad/global genomic analysis it is envisioned the second part (investigating dose response characteristics) will focus on a selected set of key marker genes mechanistically and biologically relevant to the mode of action of the test compound. In essence individual genes will not be included in the set of key marker genes unless associated with other genes within the same biological process. For example a change in a single gene associated with steroid synthesis will probably not be included in the key set unless associated with several other genes involved in the steroidogenesis pathway.
In the third part of the investigation (bioinformatics stage) all data should be rigorously mined for biomarker sets which can then be verified. All data from the investigation will be made publicly available to allow re-analysis by researchers with an interest in the investigations.
As the research will explore toxicogenomic changes across a range of doses with different periods of exposure responders are encouraged to investigate estrogenic substances with:
- existing multi-endpoint animal toxicity data base including repeat dose toxicity and developmental & reproductive investigations
- well characterized dose response characteristics, and
- characterized pharmacokinetic/toxicokinetics to link target organ dose, (rather than applied dose) with gene change and adverse outcome.
The changes in gene expression identified through this research should be able to predict future toxicity (predictive marker) with increasing dose (i.e. change from normal to adaptive) and act as diagnostic markers of toxicological change reflected in traditional toxicological endpoints (i.e. change from adaptive to adverse).
Successful applicants will be expected to present intermediate work products at appropriate scientific venues such as CEFIC LRI sponsored meetings, professional conferences and scientific advisory groups.