Status of the Glutathione Reactivity Database for Skin Sensitization

1. Status of the Glutathione Reactivity Database for Skin Sensitization T. W. Schultz Presented at the McKim Conference September 17, 2008

2. The Toxicity Pathway Framework for Predicting Hazard Speciation, Metabolism Reactivity Etc. Molecular Initiating Events In Vitro and System Effects In Vivo Adverse Outcomes Parent Chemical Up-Stream Down-Stream CHEMISTRY BIOLOGY Structure-Activity Levels of Organization

3. Hazards of Chemicals withProtein-Binding as Molecular Initiating Event • Human Health Hazards • Sensitization - skin and respiratory • Acute toxicity - inhalation gas or vapors • Chromosomal aberrations • Developmental toxicity • Selected organ-specific effects • Aquatic Toxicity • Excess toxicity for aquatic organisms

4. Mechanisms of Protein Binding • No consensus in number > 25 < 50 (40) • Vary in target moiety • Typically 45% -SH and 45% -NH2 • Vary in structural domain • Simple: isothiocyanate RN=C=S • More complex: Michael addition • Most complex: SNAr addition

5. Chemical Reactivity in Skin Sensitization Mechanisms of Protein Binding In Vitro Measurements In Chemico Measurement Hazard Assessment Endpoints In vitro effects Reactive Potency Michael addition SN2 SNAr In vivo sensitization In silico modeling

6. Relative Reactivity Different nucleophiles can differ in their absolute reactivity towards a given electrophile, but relative reactivity is well correlated over a range of nucleophiles within the same mechanism. CH2=CH- k(thiol) K(amine) ______________________________________________________ C(=O)OMe 0.0110.00076 C#N 0.0027 0.00020 C(=O)NH2 0.00046 0.000026

7. Tiered Approach to Evaluating Protein Binding • In silico; qualitative, pre-screen 1000’s of compounds • In chemico; depletion-based quantitative, screen 100’s of compounds • Adduct identification; LC-MS-MS, 10’s of compounds

8. In Chemico Reactivity • Use in a similar context to in vitro or in silico • Quantitative, rapid, inexpensive experiments with model nucleophiles • Verify mechanism-based rules of reactivity • Define the applicability domain of a reactive mechanism • Provides a measure of relative potency

9. In Chemico Thiol Reactivity Assay • Protocol (Schultz et al., 2005) is quantitative, rapid, inexpensive, abiotic, and spectrophotometric-based • Depletion method; measures % free thiol with GSH as the model nucleophile • Endpoint is RC50 (50% reactive concentration; mM) is calculated by probit analysis of concentrations-response data after 2-hrs

10. Repeatability & Reproducibility of RC50Values for Acrolein RC50 (mM) Date Lot Operator _____________________________________________________________ 0.094 02-10-05 1 A 0.081 02-18-05 1 A 0.085 02-18-05 1 B 0.086 02-19-05 1 B 0.092 03-03-05 1 A 0.074 03-06-05 1 A 0.084 03-14-05 1 A 0.085 03-21-05 1 A 0.088 12-21-06 2 C 0.068 01-03-07 2 D 0.100 01-05-07 2 D

11. + & - of GSH-BasedReactivity Assay • Readily available • Concentration can be analyzed by simple methods • Odorless, non-hazardous • Water soluble but NOT readily soluble in organic solvents • Does not lead itself to HPLC

12. Measurements of In Chemico Reactivity • Depletion of reactant or formation of product • Full Kinetics- measured at several time intervals with several initial concentrations of electrophile (100 chemicals) • Partial Kinetics- measured at several time intervals with one initial concentrations of electrophile • Concentration giving 50% reaction in a fixed time- measured at one time with several initial concentrations of electrophile (1000 chemicals) • Extent of reaction after a fixed time- measured at one time with one initial concentrations of electrophile

13. Log (RC50) versus log k

14. Group 1 Thiol –Related Mechanisms • highly relevant with complex domains • 1) Michael Addition, • 2) Nucleophilic substitution (N-sub) of haloaliphatics, • 3) N-sub of haloaromatics (SNAr).

15. Michael Acceptors Base Structure and Special Features XC=C XC#C X = -CHO, -COR, -CN, etc.

16. GSH & LLNA DATA EC3 = 0.01 RC50 = 0.03 RC50 = 0.05 RC50 = 0.05 RC50 = 0.09 RC50 = 0.02

17. Structural Variation for Potency of Esters TYPE STRUCTURE RC50 (mM) Acetylenedicarboxylates RC(=O)C#CC(=O)R 0.025 Propiolates C#CC(=O)OR 0.1 trans-Vinylene dicarboxylates RC(=O)C=CC(=O)R 0.2 Acrylates C=CC(=O)CR 0.8 cis-Vinylene dicarboxylates RC(=O)C=CC(=O)R 2.0 Methacrylates C=C(C)C(=O)CR 30.0

18. N-sub Haloaliphatic Base Structure and Special Features R1C(X)YR2 Y = C6H5 > C#C > C=C, etc X = I > Br > Cl > F R1 = H > CnH(2n +1) R2 no effect

19. N-sub Haloaromatic Base Structure and Special Features XC6H3Y2 Y = NO2, > in-ring-N > CHO > CN X = F > Cl > Br > I Position of leaving group in relationship to activity groups effects potency

20. Qualitative Read-Across for SNAr Electrophiles Substance RC50 (mM) Sensitizer _______________________________________________ 1-F-2,4-dinitrobenzene 0.07 + 1-Cl-2,4-dinitrobenzene 1.5 + 1-Br-2,4-dinitrobenzene 1.2 + 1,3-Cl-4,6-dinitrobenzene 0.24 + 1,5-Cl-2,3-dinitrobenzene 0.20 + 1,2-dinitrobenzene NRAS - 2,4-F-1-nitrobenzene NRAS - 2,4-Cl-1-nitrobenzene NRAS - 1-Br-4-Cl-2-nitrobenzene NRAS - 1,2,3-Cl-5-nitrobenzene NRAS -

21. Effect of Derivative on RC50 Values

22. Current Status of Work on Group 1Thiol –Related Mechanisms • 1) Michael Addition (largely completed), • 2) Pre-Michael Addition (nearly completed), • 3) N-sub of Haloaliphatics (largely completed), • 4) N-sub of Haloaromatics (just started).

23. Group 2Thiol –Related Mechanisms • relevant with simple domains • 1) disulfide exchange; • 2) disulfide formation, • 3) O-heterocyclic ring opening, • 4) N-sub of alkyl sulfates & sulfonates, • 5) nitroso-binding.

24. Preliminary WorkGroup 2Thiol –Related Mechanisms • 1) disulfide exchange, (5 compounds) • 2) disulfide formation, (5 compounds) • 3) O-heterocyclic ring opening, (5 compounds) • 4) N-sub of alkyl sulfates, (3 compounds) • 5) N-sub of alkyl sulfonates, (3 compounds) • 6) nitroso-binding (3 compounds). • All demonstrate GSH reactivity andare related to sensitization

25. Group 3Thiol –Related Mechanisms • less relevant • 1) arycarboxylate aminolysis, • 2) electrostatic interactions, • 3) mercury thiolate formation, • 4) oxime group condensation, • 5) others.

26. OECD QSAR Toolbox • Applies computational methods to filling data gaps • By facilitating the selection of chemical analogues and grouping chemicals into categories • Integrates (among other things) mechanisms of action with categories • Among the best characterized method, which integrates mechanisms of action into grouping chemicals is the profiler bases onProtein-binding.

27. Toolbox Protein-Binding Profiler • Currently covers 38 different mechanism • Vary in specificity • Vary in completeness • Structural Alerts for mechanisms tend to be qualitative in nature • Michael addition alerts include the extreme sensitizer p=benzoquinone, the moderate sensitizer ethyl acrylate, and the very weak sensitizer methyl methacrylate.

28. Subcategorization of Michael Acceptors by Reactivity • Extremely fast:quinones, propiolates , 1-alken-3-ones • Fast:acrylates, 2-alkenals, 3-alken-2-ones • Moderately Fast:alkyl 2-alkynoates • Slow:tiglates • Very Slow:methacryates • Non-Reactive:non-,-unsaturated

29. Application Reactivity to Catgorizing an Inventory • ≈ 1500 substances on the List of Flavor and Fragrance Related Substances • ≈1300 discrete substances of which: • 79 Fast- to moderate-reacting Michael-acceptors; • 19 Slow-reacting Michael-acceptors; • 57 Schiff-base aldehydes; • 29 Acetals; • 15 Disulfide formers; • 11 Cyclic addition diones; • 9 Disulfide exchangers; • 3 O-heterocyclic ring openers. >40 pro-electrophiles

32. Thank you.