T. W. Schultz Presented at the McKim Conference September 17, 2008

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

 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

 No consensus in number > 25 < 50 (40)  Vary in target moiety  Typically 45% -SH and 45% -NH 2  Vary in structural domain  Simple: isothiocyanate RN=C=S  More complex: Michael addition  Most complex: S N Ar addition

Michael addition S N 2 S N Ar Reactive Potency In vitro effects Mechanisms of Protein Binding In vivo sensitization Hazard Assessment Endpoints In silico modeling In Chemico Measurement In Vitro Measurements

CH 2 =CH- k(thiol) K(amine) ______________________________________________________ C(=O)OMe C#N C(=O)NH 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.

 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

 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

 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 RC 50 (50% reactive concentration; mM) is calculated by probit analysis of concentrations- response data after 2-hrs

RC 50 (mM) Date Lot Operator _____________________________________________________________ A A B B A A A A C D D

 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

 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

 highly relevant with complex domains  1) Michael Addition,  2) Nucleophilic substitution (N-sub) of haloaliphatics,  3) N-sub of haloaromatics (S N Ar).

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

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

TYPE STRUCTURE RC50 (mM) Acetylene dicarboxylates RC(=O)C#CC(=O)R 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

Base Structure and Special Features R 1 C(X)YR 2 Y = C 6 H 5 > C#C > C=C, etc X = I > Br > Cl > F R 1 = H > C n H (2n +1) R 2 no effect

Base Structure and Special Features XC 6 H 3 Y 2 Y = NO 2, > in-ring-N > CHO > CN X = F > Cl > Br > I Position of leaving group in relationship to activity groups effects potency

Substance RC 50 (mM)Sensitizer _______________________________________________ 1-F-2,4-dinitrobenzene Cl-2,4-dinitrobenzene Br-2,4-dinitrobenzene ,3-Cl-4,6-dinitrobenzene ,5-Cl-2,3-dinitrobenzene ,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 -

 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).

 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.

 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 and are related to sensitization

 less relevant  1) arycarboxylate aminolysis,  2) electrostatic interactions,  3) mercury thiolate formation,  4) oxime group condensation,  5) others.

 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 on Protein-binding.

 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.

 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

 ≈ 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

Thank you.