1. Mike Comber TIMES-SS Application of Reactivity Principles in Screening for Skin Sensitisers Presented on behalf of the TIMES-SS consortia & International QSAR Foundation

2. Outline Goal Goal – to apply the reactivity knowledge base to an economically important hazard assessment endpoint Why TIMES-SS? Why TIMES-SS? Refining TIMES-SS Refining TIMES-SS Consortium & Aims Consortium & Aims Brief overview of the model Brief overview of the model Performance & issues Performance & issues Current programme & developments Current programme & developments Future needs Future needs

3. Skin sensitisation There are in-vitro alternatives & other models….. There are in-vitro alternatives & other models….. but none are adequate for all classes of sensitisers Greater regulatory acceptance requires greater mechanistic transparency- “no black boxes” Greater regulatory acceptance requires greater mechanistic transparency- “no black boxes” Increasing regulatory acceptance Increasing regulatory acceptance Avoiding the test saves industry money! Avoiding the test saves industry money! Screening chemicals helps direct research Screening chemicals helps direct research

4. TIMES-SS :Consortium Coordination – Mike Comber – on behalf of the IQF Coordination – Mike Comber – on behalf of the IQF Research team Research team Laboratory of Mathematical Chemistry, University Bourgas Laboratory of Mathematical Chemistry, University Bourgas Dr Dave Roberts Dr Dave Roberts Consortium Consortium ExxonMobil ExxonMobil Procter & Gamble Procter & Gamble Unilever Unilever Research Institute for Fragrance Materials (RIVM) Research Institute for Fragrance Materials (RIVM) Dow Dow Danish National Food Institute Danish National Food Institute Funding & data sharing + sweat equity Funding & data sharing + sweat equity

5. Aims for TIMES-SS To develop a skin sensitisation (Q)SAR model that: To develop a skin sensitisation (Q)SAR model that: Potentially minimises the need for animal testing Potentially minimises the need for animal testing Is scientifically credible and valid to Industry and Regulatory bodies Is scientifically credible and valid to Industry and Regulatory bodies Agrees with the OECD principles for (Q)SAR validation Agrees with the OECD principles for (Q)SAR validation Mechanistically defensible Mechanistically defensible Hence has high potential for acceptance under REACH in place of animal tests Hence has high potential for acceptance under REACH in place of animal tests

6. Characterisation with the OECD principles OECD principles for (Q)SAR validation: OECD principles for (Q)SAR validation: a defined endpoint  a defined endpoint  an unambiguous algorithm  an unambiguous algorithm  a defined domain of applicability  a defined domain of applicability  appropriate measures of goodness-of-fit, robustness and predictivity  appropriate measures of goodness-of-fit, robustness and predictivity  a mechanistic interpretation  a mechanistic interpretation  where  demonstrates the concordance between TIMES and the OECD principles For full evaluations - see : Patlewicz et al., 2007 Reg Tox Pharm, 48, 225–239 & Roberts et al., 2007, Chem Res Toxicol, 20 (9), 1321–1330

7. Identifying the cause of an effect Complex toxicological endpoints are biological responses to: Complex toxicological endpoints are biological responses to: Direct molecular interactions dependent on chemical structure Direct molecular interactions dependent on chemical structure Indirect molecular interactions which are dependent on the chemical structure of metabolites Indirect molecular interactions which are dependent on the chemical structure of metabolites AND Biological processes dependent on other properties e.g. pH/chemical reactivity AND Biological processes dependent on other properties e.g. pH/chemical reactivity In TIMES-SS - trying to separate the unknowns associated with metabolism from the unknowns associated with chemical reactivity itself. In TIMES-SS - trying to separate the unknowns associated with metabolism from the unknowns associated with chemical reactivity itself. There has been a continual effort to make sure any plausible mechanism of interactions leading to a protein adduct is consistent: There has been a continual effort to make sure any plausible mechanism of interactions leading to a protein adduct is consistent: With the literature on that specific reaction mechanism, and With the literature on that specific reaction mechanism, and With the more general understanding chemical reactivity. With the more general understanding chemical reactivity.

8. Process for TIMES-SS What is the hypothesis? What is the hypothesis? How do we then model the endpoint? How do we then model the endpoint? What is the data required to then build the model? What is the data required to then build the model? What are the shortcomings and the gaps? What are the shortcomings and the gaps?

9. Epidermis Dermis Hypodermis Vein Protein conjugates Penetration Protein conjugates Metabolism Lymph Hypothesis for skin sensitization Subject of modeling Assumptions: 1.Chemicals always penetrate stratum corneum 2.Formation of protein conjugates is a premise for ultimate effect 3.Metabolism may play significant role in skin sensitization

10. Modeling the hypothesis Modeling skin metabolism Modeling skin metabolism Skin metabolic simulator contains 336 hierarchically ordered spontaneous and enzyme controlled reactions. Skin metabolic simulator contains 336 hierarchically ordered spontaneous and enzyme controlled reactions. Covalent interactions of chemicals/metabolites with skin proteins are described by 67 alerting groups. Covalent interactions of chemicals/metabolites with skin proteins are described by 67 alerting groups. 3D-QSARs are applied for some of these alerting groups to improve the associated predictability. 3D-QSARs are applied for some of these alerting groups to improve the associated predictability.

11. Different type of principal transformations Phase I reactions Ester hydrolysis Schiff base formation with aldehydes Phase II reactions Glucoronidation Interactions with proteins Nucleophilic substitution on halogenated C sp3 atom

12. Protein binding alerts requiring 3D QSAR (COREPA) models 1. Aldehydes R = alkyl, H 2. α,β- unsaturated carbonyl compounds acting by Michael addition R = OC, C, N, S

13. What is the data required to then build the model? Training set of 885 chemicals with experimental data from three sources (LLNA, GPMT, BfR). Training set of 885 chemicals with experimental data from three sources (LLNA, GPMT, BfR). Experimental skin sensitisation data Experimental skin sensitisation data Reaction data – data on reactivity is not available. Discrimination by chemical reactivity is semi qualitative Reaction data – data on reactivity is not available. Discrimination by chemical reactivity is semi qualitative

14. Ranges of chemical reactivity are introduced - Examples Nucleophilic acyl substitution in azalactones causing Strong skin sensitization effect Chemicals from the training set having observed Strong sensitization effect

15. Nucleophilic acyl substitution in azalactones causing Weak skin sensitization effect Chemicals from the training set having observed Weak sensitization effect Ranges of chemical reactivity are introduced – Examples (Contd.)

16. Michael addition on a,b - aldehydes causing Strong skin sensitization effect Chemicals from the training set having observed Strong sensitization effect Ranges of chemical reactivity are introduced – Examples (Contd.)

17. Michael addition on a,b - aldehydes causing Weak skin sensitization effect Chemicals from the training set having observed Weak sensitization effect Ranges of chemical reactivity are introduced – Examples (Contd.)

18. Current shortcomings and needs The level of support for reactions is variable The level of support for reactions is variable Some reactions have 1 supporting chemical Some reactions have 1 supporting chemical New reactions are being generated as new data is reviewed – requiring further assessments New reactions are being generated as new data is reviewed – requiring further assessments Reactivity data for the reactions/chemical groups is not available Reactivity data for the reactions/chemical groups is not available A semi qualitative potency scale is used for some of the reactions/chemical groups A semi qualitative potency scale is used for some of the reactions/chemical groups