1. 11 Simulating of in vivo metabolism taking into account detoxification logics

2. 12 AMES CA Liver Bone Marrow Blood Transport Activation (Phase I) Conjugation (Phase II) DNA/Protein reactivity no pharmacokinetics factors Activation (Phase I) Conjugation (Phase II) DNA/Protein reactivity pharmacokinetics factors in vivo detoxification “logic” Effect in vivo bio-exhausting in vitro Genotoxicity in vivo Genotoxicity Levels of GT Investigation

3. 13 Levels of GT Investigation: First-Pass Metabolism? in vivo liver genotoxicity in vivo MN genotoxicity negative Level ILevel II Level III in vitro mutagenicity negative positive negative (metabolic detoxification in liver) positive negative (bio-exhausting) positive chemical

4. 14 Levels of GT Investigation: First-Pass Metabolism? in vivo liver genotoxicity in vivo MN genotoxicity negative Level ILevel II Level III in vitro mutagenicity negative positive negative (metabolic detoxification in liver) positive negative (bio-exhausting) positive

5. 15 Levels of GT Investigation: First-Pass Metabolism? in vivo liver genotoxicity in vivo MN genotoxicity negative Level ILevel II Level III in vitro mutagenicity negative positive negative (metabolic detoxification in liver) positive negative (bio-exhausting) positive

6. 16 Simulating of in vivo metabolism taking into account detoxification and bio-exhausting

7. 17 Simulating of in vivo detoxification  simulating in vivo detoxification  simulating in vivo bio-exhausting

8. 18 Simulating of in vivo detoxification  simulating in vivo detoxification  simulating in vivo bio-exhausting

9. 19 In vivo Detoxification Includes:  Principal phase II metabolic detoxification reactions:  glutathione conjugation  glucuronidation  amino acid conjugation  acetylation  sulfation  Detoxification “logic” (Complete metabolic detoxification suppresses genotoxic action of reactive intermediates in liver).

10. 110 In vivo Detoxification of Styrene (in vivo metabolism)

11. 111 R=Aryl DNA reactivity observed in vitro Protein binding observed in vitro In vitro genotoxic effects of styrene epoxide Styrene oxide is hydrolyzed in vitro to styrene glycol by microsomal epoxide hydrolase from the liver, kidneys, intestine, lungs, and skin of several mammalian species (Oesch 1973, cited in IARC 1985). Observed in vitro metabolic pathway for styrene

12. 112 Mutagenic effects of styrene in vivo can be expected under extreme exposure conditions if styrene oxide is not efficiently detoxified and primary DNA lesions are not completely repaired. (Speit et al. 2008). R=Aryl DNA reactivity observed in vitro Protein binding observed in vitro “Trapped” metabolite due to the “channeling” effect Observed in vivo MNT metabolic pathway for styrene

13. 113 Observed in vivo MNT metabolic pathway for styrene A recent published data for in vivo MNT in bone marrow cells of mice was clearly negative (Speit et al. 2008). R=Aryl DNA reactivity observed in vitro Protein binding observed in vitro “Trapped” metabolite due to the “channeling” effect

14. 114 Simulating in vivo detoxification of styrene by TIMES

15. 115

16. 116 “Trapped” reactive metabolites in in vivo detoxification Pathway I

17. 117 “Trapped” reactive metabolites in in vivo detoxification Pathway II

18. 118 Implementation of Detoxification “Logic” Related to In Vivo Bone Marrow MNT Test Results – Classes of Chemicals Studied  Aromatic amines  Organic halides  Nitro compounds  Epoxides  Ureides  Isocyanates

19. 119  Aromatic amines  Organic halides  Nitro compounds  Epoxides  Ureides  Isocyanates Example: Preventing in vivo N-Hydroxylation Y = –SO 3 H, -COOH, COOR, -P(=O)(OH) 2, phosphate, thiopohosphate, etc. Observation: Polar functional groups in aromatic amines prevent phase I N-hydroxylation as bioactivation reaction. Reasoning:  the chemicals are already polar enough to be subjected to phase II detoxification reaction;  electron-withdrawing functional groups hamper the aromatic amine N-hydroxylation Implementation of Detoxification “Logic” Related to In Vivo Bone Marrow MNT Test Results – Classes of Chemicals Studied

20. 120 Simulating of in vivo detoxification  simulating in vivo detoxification  simulating in vivo bio-exhausting

21. 121 The in vivo bio-exhausting detoxification scenarios include: highly reactive metabolites of liver GT chemicals are bio-exhausted approaching to the MN bone marrow due to off-target reactions, therefore, they become incapable of producing harmful effects on the target tissue (bone marrow). bio-exhausting of short – lived intermediates formed in liver Bio-exhausting Detoxification Scenarios:

22. 122 Negative in vivo GT (MNT) effect of Nitrobenzene

23. 123 Negative in vivo GT (MNT) effect of Nitrobenzene “Trapped” reactive metabolites in in vivo detoxification Pathway I Liver genotoxic metabolite is further bio-exhausted along its path to the bone marrow

24. 124 Negative in vivo GT (MNT) effect of Nitrobenzene “Trapped” reactive metabolites in in vivo detoxification Pathway II Liver genotoxic metabolite is further bio-exhausted along its path to the bone marrow

25. 125 Negative in vivo GT (MNT) effect of Nitrobenzene “Trapped” reactive metabolites in in vivo detoxification Pathway III Liver genotoxic metabolite is further bio-exhausted along its path to the bone marrow