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 with Protein-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
Michael
addition
SN2
SNAr
Reactive
Potency
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
C#N
C(=O)NH

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 RC50 Values for Acrolein
RC50 (mM) Date Lot Operator
_____________________________________________________________ A A B B A A A A C D D

11. + & - of GSH-Based Reactivity 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. Base Structure and Special Features
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

17. Structural Variation for Potency of Esters
TYPE STRUCTURE RC50 (mM)
Acetylene dicarboxylates RC(=O)C#CC(=O)R
Propiolates C#CC(=O)OR
trans-Vinylene dicarboxylates RC(=O)C=CC(=O)R
Acrylates C=CC(=O)CR
cis-Vinylene dicarboxylates RC(=O)C=CC(=O)R
Methacrylates C=C(C)C(=O)CR

18. Base Structure and Special Features
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. Base Structure and Special Features
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
1-Cl-2,4-dinitrobenzene
1-Br-2,4-dinitrobenzene
1,3-Cl-4,6-dinitrobenzene
1,5-Cl-2,3-dinitrobenzene
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 1 Thiol –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 2 Thiol –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 Work Group 2 Thiol –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 and are related to sensitization

25. Group 3 Thiol –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 on Protein-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.