Grouping and Read-Across for Respiratory Sensitisation. Dr Steve Enoch School of Pharmacy and Biomolecular Sciences Liverpool John Moores University

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1 Grouping and Read-Across for Respiratory Sensitisation Dr Steve Enoch School of Pharmacy and Biomolecular Sciences Liverpool John Moores University

2 Chemicals are grouped into a category Toxicity data from the category members are used to predict the target chemical

3 Respiratory Sensitisation A range of LMW organic chemicals can cause respiratory sensitisation e.g. isocyanates No suitable animal or in-vitro model limited data Majority of reported respiratory sensitisers are from human case studies LLNA used in risk assessment however not all skin sensitisers are also respiratory sensitisers

4 Biological Mechanism Single type of immune response: Th2 In skin sensitisation the Th2 response has been suggested to involve covalent bond formation with nitrogen The lung is an oxidising environment removing (or limiting) the number of free thiol groups Therefore, likely that lysine is the nucleophile

5 Skin Versus Lung Exposure We know that induction of respiratory sensitisation can occur either in the lung or through the skin The human respiratory sensitisation data we have access to is derived from exposure in the lung It is important to realise that the chemistry of induction and elicitation is the same (regardless of whether it occurs in the skin or the lung)

6 Mechanistic Profiling Parent Chemical Speciation, Metabolism Reactivity Etc. Molecular Initiating Event (MIE) Cellular and System Effects In Vivo Adverse Outcomes Up-Stream CHEMISTRY Structure-Activity Down-Stream BIOLOGY Levels of Organisation Covalent binding to a protein

7 Development of an In Silico Profiler Profiling: The ability to identify a potential Molecular Initiating Event (MIE) within a target chemical Category formation: Identify chemicals from a database that can undergo the same MIE Sensitisation: one MIE is covalent protein binding

8 Development of an In Silico Profiler A profiler contains structural alerts related to the MIE It is created by analysing data related to the MIE this can be toxicological or chemical A profiler DOES NOT contain information that can be used to distinguish active compounds from inactive It ONLY contains information related to the MIE

9 Respiratory Sensitisation: Clinical Data 104 chemicals each with a molecular weight less than 1000 g/mol All data taken from clinical reports Data from previous studies covered in this dataset (65 additional chemicals) These data are not ideal; however, they are what are currently available

10 Structural Alert Examples We had previously identified 22 structural alerts associated with respiratory sensitisation

11 Initial Profiler Analysis Structural alert No structural alert Respiratory sensitisers (104) Importantly, the absence of an alert gives no indication about whether a chemical is a sensitiser or not Evidenced by the number of sensitising chemicals that do not contain an alert There will ALWAYS be areas of chemical space that have not been explored that MIGHT contain new structural alerts

12 Initial Profiler Analysis Structural alert No structural alert Respiratory sensitisers (104) Importantly, the absence of an alert gives no indication about whether a chemical is a sensitiser or not Evidenced by the number of sensitising chemicals that do not contain an alert There will ALWAYS be areas of chemical space that have not been explored that MIGHT contain new structural alerts

13 Schiff Base Formation: Methanamine

14 Michael Addition: Azocarbonamide Michael addition Respiratory sensitisation

15 Michael Addition: Methyl Tiglate Michael addition X No Respiratory sensitisation Example of the need for a chemical to pass the electrophilicity threshold to cause respiratory sensitisation

16 S N 2: Chlorhexidine N-chlorination (as little as 2 ppm) S N 2 Respiratory sensitisation It is important to consider the exposure route - alert is only applicable to exposure around swimming pools

17 S N 2: 3-Amino-5-mercapto-1,2,4-triazole In this example the chemical is acting as the NUCLEOPHILE and the di-sulfide bridge is the ELECTROPHILE (the dataset contained 4 examples of this type of mechanism) Recent work has shown this mechanism to also be important for skin sensitisation

18 Irritants: Carboxylic Acids Frequently reported in the clinical data as being respiratory sensitisers This seems unlikely based on their chemistry with it being more likely that they acts as irritants to the respiratory tract

19 Profiler Summary Mechanistic domain Number of alerts Acylation 7 Michael addition 16 Schiff base formation 16 S N 2 9 S N Ar 4 Radical 1 Total 53

20 Structural Alert Usage The structural alerts in the profiler are related to covalent protein binding in the lung for LMW organic chemicals They are intended for category formation and/or hazard identification (prioritisation) Importantly, the absence of an alert gives NO information about toxicity

21 Electrophilicity Threshold Low electrophilicity No cross-linking Low electrophilicity Protein cross-linking High electrophilicity No cross-linking

22 Read-Across for Azocarbonamide = 1.45 ev Assigned domain: Michael addition = 1.24 ev Non-sensitiser = 1.72 ev Sensitiser = 2.18 ev Sensitiser

23 Read-Across for Methyl Tiglate = 1.18 ev Assigned domain: Michael addition = 1.24 ev Non-sensitiser = 1.72 ev Sensitiser = 2.18 ev Sensitiser

24 Electrophilicity The rate of covalent bond formation is important in determining potency in skin sensitisation This is likely to be true for respiratory sensitisation In chemico data are becoming available

25 An MIE Knowledge Base Protein binding is the key MIE for low molecular weight organic chemicals for both skin and respiratory sensitisation Structural alerts are typically derived from analysis of toxicological datasets This leads to a limited coverage of the potential chemical space

26 An MIE Knowledge Base Gold standard of direct acting structural alerts Define organ specific deactivating features Define organ specific activating features Organ specific profiler Chemotype development - extended structural alerts that incorporate key physico-chemical properties such as: Electrophilicity, Volatility, Hydrophobicity

27 Schiff Base Formation: Methanamine

28 Schiff Base Formation: Formaldehyde Releasers

29 Other MIEs We know a lot about electrophiles Focus has been on analogous mechanisms to skin sensitisation Other MIEs that lead to respiratory sensitisation? High molecular weight compounds Radicals/metals

30 AOPs and In Silico Profiling In silico profilers are typically focussed around the MIE This is useful for grouping and prioritisation Currently, these are the best methods with which to make predictions However, such predictions need support from other assays focussed around key events in an AOP

31 Conclusions Analysis of respiratory sensitisation data has shown covalent protein binding to be a key MIE allowing an in silico profiler to be developed These structural alerts are suitable for category formation, hazard identification and prioritisation Electrophilicity, cross-linking and volatility are important factors in determining sensitisation potential In vitro assays relating to the other key events in the AOP are going to be needed to support the in silico knowledge around the MIE

32 Key References Respiratory sensitisation Enoch et al (2009) Chem Res Toxicol, 22, p1447 Enoch et al (2010) Chem Res Toxicol, 23, p1547 Enoch et al (2012) Chem Res Toxicol, 25, p2490

33 Acknowledgements University of Manchester Dr Martin Seed Dr Raymond Agius Liverpool John Moores University Dr Dave Roberts The research leading to these results has received funding from the European Community s Seventh Framework Program (FP7/ ) under grant agreement n and from Cosmetics Europe.