ILSI HESI Technical Committee on the Application of Genomics to Mechanism Based Risk Assessment

1. ILSI Health and Environmental Sciences Institute ILSI HESI Technical Committee on the Application of Genomics to Mechanism Based Risk Assessment

2. Challenges Facing Future Application of Toxicogenomics • Lack of publicly available databases • Lack of validation of available technologies • Lack of comparable tools, methods, study designs • Lack of robust tools for data analysis • Lack of knowledge – how transcription products relate to toxicity. Relevance of single gene changes. • Uncertain regulatory applications / environment

3. ILSI Health and Environmental Sciences Institute ILSI Health and Environmental Sciences Institute Non-profit research and educational organization Provides an international forum for scientific activities Participation from industry, government, academia Research programs, workshops, databases, expert panels, publications Reputation for focus and objectivity

4. ILSI Health and Environmental Sciences Institute Committee Overview Committee formed in 1999 Membership: ~30 Companies – Pharmaceutical, Consumer-Product, Chemical, and Agricultural Chemical International (US, Europe, Japan) Government Participation (NIEHS, NCI, NIH, NCTR, U.S. FDA, US Dept of Army, US Air Force, and EPA, Japan NIHS) Academic Participation (U. Surrey (UK), Michigan State University, Medical College of Wisconsin, TIGR)

5. ILSI HESI Genomics Committee Objectives • Evaluate experimental methodologies for measuring alterations in gene expression • Develop publicly available data • Contribute to development of public international databases linking gene expression data and key biological parameters • Determine if known mechanisms and pathways of toxicity can be associated with characteristic gene expression profiles and if this information can be used for risk assessment.

6. Committee Initiated 1999 2000 2001 2002 2003 2004 2005 Hepatotoxicity Nephrotoxicity Genotoxicity Database Work Groups Formed HESI Committee on Genomics ILSI HESI- EBI Dbase Collaboration Start Public Mtg on Genomics in RA Experimental Program Initiated Peer-Reviewed Publications Experimental Program • ~30 Members from Pharma, Ag Chem, Consumer Product, and Chemical Industries • NIEHS, NCI, NIH, NCTR, U.S. FDA, US Dept of Army, US Air Force, and EPA, Japan NIHS + Academics

7. Committee on theApplication of Genomics in Mechanism Based Risk Assessment ILSI Health and Environmental Sciences Institute Program Mechanism Toxicology and Microarrays Toxicity Working Groups Hepatotoxicity Nephrotoxicity Genotoxicity Steering Team Database Database Working Group Collaboration with EBI

8. Common Experimental Design Features • Well-studied compounds - known toxicity profiles/biological parameters • Investigate temporal relationships • Low vs. high dose response relationships – multiple dose levels • Variability – how many replicates needed? • Compare SOPs from lab to lab • Compare different types of platforms

9. ILSI HESI Genomics Committee Objectives Objective 1: Evaluate experimental methodologies for measuring alterations in gene expression • Develop protocols to evaluate toxicity profiles of several prototypic genotoxicants, hepatotoxicants and nephrotoxicants - completed • Distribute RNA samples to public and industry labs for microarray-based gene expression analysis - completed • Evaluate influence of specific experimental conditions on data variability – in progress • Utilize outcome of experiments to stimulate discussion of “best practices” – in progress

10. ILSI HESI Genomics Committee Objectives Objective 2: Contribute to development of international databases linking gene expression data and key biological parameters • Build short-term gene expression data repository for well characterized toxicants with known mechanisms of action under defined experimental conditions – completed (ILSI Microarray Database) • Consider appropriate linkages to experimental and biological data – in progress with EBI • Utilize experience to contribute to international discussion on appropriate database features and structure – in progress with EBI and others • Make database available in the public domain – scheduled for 2004

11. ILSI HESI Genomics Committee Objectives Objective 3: Determine if known mechanisms and pathways of toxicity can be associated with characteristic gene expression profiles and if this information can be used for risk assessment. • Develop toxicity and gene expression data sets on well characterized toxicants with known mechanisms of action under defined experimental conditions - completed • Evaluate gene expression profiles and relate to other measures of toxicity - completed • Use information to identify potential biomarkers and interspecies extrapolation issues – under consideration as next step • Help build consensus on optimal approaches for interpretation of gene expression data and its application to risk assessment – in progress

12. ILSI Health and Environmental Sciences Institute Nephrotoxicity Working Group (3 compounds) In-life Study (1 in-life lab per compound) Participating Laboratories (~8) RNA • Microarray Analysis • Inter- and Intra-lab variability • Cross-platform variability • Replicate In-life study RNAs/cDNA Analyzed On Multiple Platforms (Affymetrix, Incyte, ClonTech, Phase-1, NIEHS, custom)

13. Nephrotoxicity Working Group – Interim Findings • NOEL and expected toxicity achieved. • Transcriptional analysis yielded strong topographic specificity and some mechanistic information. • All confirmatory analyses were positive and will be extended to investigate potential biomarkers of nephrotoxicity. • Frequency of individual animal transcript changes was reduced in nonresponders and increased in cases of severe toxicity. • Use of pooled RNA samples may have a dilution/skewing effect on interpretation of genetic response. • At least equal sensitivity of microarray technology to traditional toxicology endpoints.

14. ILSI Health and Environmental Sciences Institute Hepatotoxicity Working Group (2 test compounds) In-life Compound A Study- Lab A In-life Compound A Study - Lab B Participating Laboratories (14) RNA • Microarray Analysis • Inter- and Intra-lab variability • Cross-platform variability • Replicate In-life study RNAs/cDNA Analyzed On Multiple Platforms (Affymetrix, Incyte, ClonTech, Phase-1, MD, NIEHS, custom)

15. Hepatotoxicty Working Group – Interim Findings • Expected outcome – histopathology, clinical chemistry • Clofibrate: increased liver weight, centrilobular hypertrophy, changes in alkaline phosphatase, lipid metabolism, increase in AcylCoA oxidase activity • Methapyrilene: minimal portal infiltrate, degeneration, necrosis, increased serum AST, ALT and ALP • Within a given platform (Affymetrix) there was a high degree of concordance (>90%) in the direction of the fold change across samples analyzed in different laboratories. • Lesser concordance was observed when identifying probe sets that were regulated above or below a certain threshold for all data sets (e.g., > 4 fold up-regulation). This result may be attributable to differences in data capture algorithms across laboratories. • Dose-related responses were observable in the transcriptional analysis. • For methapyrilene, agreement was found across all platforms (Affymetrix, membrane, or glass slide cDNA platforms used at a total of 10 different sites) with good but varying degrees of congruence in the results.

16. ILSI Health and Environmental Sciences Institute • Genotoxicity Working Group Mouse lymphoma (p53-) Human TK6 (p53+) Standard Genotox Protocol 14 Test Compounds (direct acting mutagens and clastogens) RNA/cDNA • Inter and Intra-lab Variability • Study Replicates • Comparison to Standard • Gene-tox assay sensitivity • ID fingerprint distinguishing • Direct from indirect acting Microarray Analysis on Own Samples RNA or cDNA Analyzed On Multiple Platforms (Affymetrix, Incyte, ClonTech Phase-1, MD)

17. Genotoxicity Working Group – Interim Findings • Gene changes < 3 fold common in studies even at highly genotoxic concentrations. • Concerns about oversensitivity of technology are likely unfounded. • Array technology may not be as sensitive an endpoint as traditional genotox assays. • Gene expression changes may distinguish between mechanistic classes of genotoxic compounds. • Standardization of analysis and control of experimental variables pose challenges to data comparison and interpretation.

18. Committee-wide Data Findings • Usual sources of variability associated with toxicology studies were notedPLUS: • mRNA processing in different labs (even w/ same protocol) • Hybridization (cDNA vs. oligo effect) • Annotation errors in various platforms • Different sequences w/in probe sets across platform (detecting different splice variants) • Presence/absence of gene on array • Effect of signal to noise ratios (in vitro) • Analysis settings (e.g., PMT) • False positives/ false negatives • Use of different analytical tools

19. Genotox Hepatotox Nephrotox

20. Database Development • Ongoing collaboration with European Bioinformatics Institute (EBI) • Building on ArrayExpress Database • House array data from multiple platforms • MIAME compliant • Adding a linked toxicology component • HESI, NIEHS, FDA, EBI, academics - new MIAME-Tox • Analysis across working groups and array platforms when completed. • Completion and population scheduled for mid 2003 – database to be made public in 2004

21. Array design • Hepatotoxicity • Nephrotoxicity • Genotoxicity Part1 Part 2 Login Create account Submission type Protocols Experiment Part 1 In-vivo Pending or New Experiment Sample description, and biological findings Experiment design Sample n Sample 1 Sample 2 Sample 4 Extraction Aliquot and pools Extracts 1….n Extracts 1….n Extracts 1….n Extracts 1….n Part 2 (Array) Labeled Extracts 1….n Labeled Extracts 1….n Labeled Extracts 1….n Labeled Extracts 1….n Array 1….n Array 1….n Array 1….n Array 1….n Hybridisations 1….n Hybridisation Data 1….n Data 1….n Data 1….n Data 1….n Scanning Combined Experiment Data Image analysis Submit Data files Transformation Final data file Labeling Array name Sample protocols

22. ILSI Health and Environmental Sciences Institute Program Status, 2003Actions • Completing data analysis on results from current studies (first quarter 2003), publication strategy (across the initiative) through 2003. • Completed interim review • Invitational workshop to discuss interpretation and application of Genomic data in risk assessment (2003) • Collaborations to analyze issues of variability. Internal efforts within and across participant laboratories Collaboration on platform issues (Affymetrix) Collaboration with data analysis experts (Rosetta) • EBI Database development and population ongoing. • MIAME-like standard for Toxicogenomics under discussion for development between NCT, ILIS and EBI (for discussion / proposal at MGED meeting Denver Feb 03)

23. ILSI Health and Environmental Sciences Institute Timeline & Next Steps • White papers on interim findings - http://hesi.ilsi.org/ in late February 2003 • Peer-reviewed publications in Spring 2003 • Invitational Workshop with Regulatory Community and other Guests– June 2003 • EBI Database population – ongoing • Ongoing discussion on applications of genomic data to risk assessment and best practices

24. Opportunities • Unprecedented opportunity to compare multiple platforms, analysis methodologies and inter-laboratory variability issues. • Opportunity to share best practice on protocols and technical issues. • Opportunity to engage database expertise and to “seed” a publicly-accessible and linkable database and to ensure such a database is able to incorporate or link to toxicological information. • Opportunity to contribute to discussions on appropriate regulatory applications of the technology; based on shared experience rather than perception. • Opportunity to promote appropriate usage in an industrial setting to maximize the usage of these approaches in a holistic safety assessment process.