Terry Schultz Professor Emeritus, The University of Tennessee-Knoxville & Secretariat, OECD, Paris

1. An Evaluation of the OECD QSAR Toolbox Profilers for Identifying DNA Reactive and Genotoxic Chemicals Terry Schultz Professor Emeritus, The University of Tennessee-Knoxville & Secretariat, OECD, Paris

2. Outlook • The Toolbox • 1st Exercise: Goal, Methods & Results • 2nd Exercise: Goal, Methods & Results • 3rd Exercise: Goal, Methods & Results • Conclusions

3. The OECD QSAR Toolbox The seminal feature of the Toolbox is its ability to quickly evaluate all members of a category for common toxicological behaviour or consistent trends within important regulatory endpoint data. This is done by first profiling the chemical by type, class or defined hazard identifiers and then selecting a category or subcategory. This feature often links the chemicals in the category to a single mechanism or mode of action.

4. Advantages of the Profile/Category Approach • it shifted emphasis to intrinsic chemical activity, • it allows for entire categories of chemicals to be assessed when only a few members are tested, • it allows for filling data gaps using read-across and trend analysis, and not just (Q)SAR models, and • it enables defensible hazard assessment through mechanistic comparisons without testing.

5. 1st McKim Workshop on Reducing Data Redundancy in Cancer Assessment The participants agreed that chemical which are QSAR predicted to be DNA reactive (+) and are also AMES test positive (+) would be chemicals of “very high concern” for the Rodent Cancer Assay. But you can have direct or indirect DNA-reactivity and AMES positive w/o S9 and w/ S9. So the first exercise was to look for members of the direct DNA-reactivity and AMES positive w/o S9 category.

6. General Flow Diagram for Screening Large Inventories for Carcinogenicity Inventory Protein OASIS Y Chrom Ab ? MicroNucl ? N Direct DNA reactive Ames Positive w/o S9 Y Bacterial Mutagen In vivo Mammal Tests Y N Generate metabolites Y Y Indirect DNA reactive Ames Positivewith S9 Refine TIMES/ Structural alerts Chrom Ab ? MicroNucl ? N Return for further screening Protein Reactive Oxidative stress? Receptor-Based Epigenetic Screen Low Carcinogenicity Potential

7. Focus Area of this Discussion Inventory Protein OASIS Y Chrom Ab ? MicroNucl ? N Direct DNA reactive Ames Positive w/o S9 Y Bacterial Mutagen In vivo Mammal Tests Y N Generate metabolites Y Y Indirect DNA reactive Ames Positivewith S9 Refine TIMES/ Structural alerts Chrom Ab ? MicroNucl ? N Return for further screening Protein Reactive Oxidative stress? Receptor-Based Epigenetic Screen Low Carcinogenicity Potential

8. Toolbox Work Flow Chemical input Profiling Endpoints Category Definition Filling data gap • QSAR Toolbox profiles for DNA binding • by OASIS • by OECD Report OASIS databasefor AMES mutagenicity Direct DNA Reactive & Ames Positive w/o S9

9. Factors Which Effect Makeup of Chemical Category How one defines the initial target chemical. For Example: Using CH4 as the initial target chemical and profiling for chemicals containing a C-atom one retrieves 8684 chemicals from the Toolbox. Whether one used DNA-binding OASIS or DNA-binding OECD. This impacts the number and definitions of the SAs which are fired.

10. Initial Database Inventory Protein alerts? 2832 Y Y Chrom Ab ? MicroNucl ? N Direct DNA reactive Ames Positivew/o S9 Y N Bacterial Mutagen In vivo Mammal Tests Y N Generate metabolites Y Y Indirect DNA reactive Ames Positive with S9 Refine TIMES/ Structural alerts Chrom Ab ? MicroNucl ? N Return for further screening Protein Reactive Oxidative stress? Receptor-Based Epigenetic Screen Low Carcinogenit Potential

11. OASIS Direct DNA Binder in Initial Database Inventory Protein alerts? 2832 Y Y Chrom Ab ? MicroNucl ? N Direct DNA reactive Ames Positive w/o S9 Y 745 N Bacterial Mutagen In vivo Mammal Tests Y N Generate metabolites Y Y Indirect DNA reactive Ames Positive with S9 Refine TIMES/ Structural alerts Chrom Ab ? MicroNucl ? N Return for further screening Protein Reactive Oxidative stress? Receptor-Based Epigenetic Screen Low Carcinogenit Potential

12. OASIS Direct DNA Binders & Ames Positive w/o S9 in Initial Database Inventory Protein alerts? 2832 Y Y Chrom Ab ? MicroNucl ? N Direct DNA reactive Ames Positivew/o S9 Y 745 N Bacterial Mutagen In vivo Mammal Tests Y N 307 Generate metabolites Y Y Indirect DNA reactive Ames Positive with S9 Refine TIMES/ Structural alerts Chrom Ab ? MicroNucl ? N Return for further screening Protein Reactive Oxidative stress? Receptor-Based Epigenetic Screen Low Carcinogenit Potential

13. OASIS (+/+) category 134 28 25 24 19 17 16 15 13 12 8 8 7 6

14. DNA-binding OECD Eliminated: Nitro Aromatic Compounds, Aromatic Amines, PAHs etc. Classes where it is unclear that a bacterial test would mimic the reactions well.

15. 2nd Exercise Designed to look for which members of the +/+ category have been evaluated in a Rodent Cancer Assay (RCA) and to report what classes they represent.

16. Toolbox Work Flow Chemical input Profiling Endpoints Category Definition Filling data gap Report CPDB Carcinogenic Potency Data Base and/or ISSCAN Carcinogenicity & mutagenicity data base Direct DNA Reactive & Ames Positive w/o S9

17. RCA for +/+ Category Inventory Protein alerts? Y Y Chrom Ab ? MicroNucl ? N Direct DNA reactive Ames Positivew/o S9 Y N Bacterial Mutagen In vivo Mammal Tests Y N 307 Generate metabolites Y Y Indirect DNA reactive Ames Positive with S9 53 Positive 11 Negative 243 No data Refine TIMES/ Structural alerts Chrom Ab ? MicroNucl ? N Return for further screening Protein Reactive Oxidative stress? Carcinogenicity Data Receptor-Based Epigenetic Screen Low Carcinogenit Potential

18. Carcinogenicity of 307 +/+ chemicals 134 28 25 24 19 17 16 15 13 12 8 8 7 6

19. 3rd Exercise Designed to look at which DNA-reactions are represented by the (+/+) chemicals that are also positive in the RCA.

20. Major Classes Aliphatic Halides [CX]; 17 CRA positive; 2 CRA negative (1-chlorobutane; 2-chloroethanol) Stressed Hetero-Ring Systems (e.g. C1CO1); 13 CRA positive

21. Minor Classes SN2 addition at an sp3 carbon atom sulfates and sulfonates; 4 CRA positive Phosphorates; 2 CRA positive Hydazines; 1 CRA positive

22. Summary In this discussion we used a hypothesis testing scheme with the QSAR Toolbox; the DNA-binding profiler generate the initial hypothesis and an in vitro test (AMES testing w/o S9) tested the hypotheses.

23. Summary We maintain that chemicals that pass this hypothesis testing are “chemicals of high concern” for cancer.

24. Summary We then asked which of these chemicals of high concern have been tested in the RCA? What are the DNA reactions covered by this suite of RCA-tested chemical?

25. Conclusion There is experimental evidence that there are opportunities to reduce the use of RCA, especially for chemicals with an aliphatic halide or stressed hetero-ring sub-structure.