Skin sensitisation prediction using in vitro screening methods Chin Lin Wong B. Biotechnology (Hons) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2015 School of Pharmacy [This page intentionally left blank] Abstract My PhD research project was designed to bring innovation into in vitro methods for identification of industrial chemicals, particularly epoxy resin systems (ERS), with skin sensitising potential as a means to minimise the use of animals for this purpose. In my research, the generalisability of two in vitro methods originally developed and validated for identification of small molecules to assess the skin sensitising potential of ERS was assessed. Specifically, my research focussed on (i) the human cell line activation test (h- CLAT) which mimics the characteristics of Langerhans cells during the maturation process following their activation by chemical sensitisers, and (ii) the direct peptide reactivity assay (DPRA), that assesses the initial interaction between potential chemical sensitisers with human skin proteins. The ERS data generated using these in vitro methods were compared with the skin sensitisation data for the same compounds generated using the widely accepted murine local lymph node assay (LLNA) in order to gain insight into the accuracy and reliability of the in vitro methods. For h-CLAT, I optimised the assay conditions for a 96-well format using 1.6x105 cells/well as well as anti-CD54-FITC and anti-CD86-PE. The relative fluorescent intensity (RFI) of CD54 and CD86 on THP-1 cells was determined using three-coloured flow cytometry. A chemical was regarded as being a positive sensitiser if the RFI of CD54 was >200% and/or that for CD86 was >150%. Five ERS tested in the h-CLAT assay, viz bisphenol A diglycidyl ether (DGEBA), trimethylolpropane triglycidyl ether (TMPTGE), poly(ethyleneglycol) diglycidylether (PEGGE) tetraphenylolethae glycidyl ether (THETGE), and poly[(phenyl glycidyl ether)-co-formaldehyde] (PPGE) gave negative results. These findings imply that the h-CLAT assay undertaken in standard format may be unsuitable for assessing skin sensitisation potential of ERS as the CD54 and CD86 were not induced. To address this issue, I investigated the possibility that ERS induced cytokine release in the h-CLAT assay may be a more sensitive marker of skin sensitisation than changes in expression levels of CD54 and CD86 on THP-1 cells. Encouragingly, concentrations of the cytokines, IL-6 and IL-8, in the cultured THP-1 cell supernatant quantified using a Meso ScaleTM Discovery human pro-inflammatory multiplex immunoassay, were markedly increased in DGEBA, TMPTGE, THETGE and PPGE. For the DPRA, chemicals with known sensitising capacity were incubated with three synthetic heptapeptides, Cor1-C420 (Ac-NKKCDLF), heptapeptides containing cysteine i (Ac-RFAACAA) and lysine (Ac-RFAAKAA) in order to determine the optimal experimental conditions. The sensitising potential of the chemicals were correlated with depletion of each heptapeptide individually in a reaction mixture. The applicability of the DPRA to assess the skin sensitising potential ERS was investigated together with known positive and negative control compounds. The aforementioned heptapeptides were selected as they had been previously shown to have a high correlation with LLNA data when used to assess small molecules. My DPRA findings show that the optimal incubation temperature for incubation of all heptapeptides was 25°C. Importantly, my data also show that the apparent heptapeptides depletion level is affected by the tube materials used for the DPRA. Specifically, Cor1- C420 was stable in polypropylene tubes but failed to meet the assay acceptance criteria for days 1-3 when borosilicate glass tubes were used. As for cysteine, it was not stable on day 3 post-incubation when glass was used for the assay. Although lysine was stable in both polypropylene and glass tubes during the course of the DPRA, the apparent extent of lysine depleted by the chemical, ethyl acrylate, differed between polypropylene (24.7 ± 5.8%) and glass (47.3 ± 7.7%) vials. Another novel finding was instability of the peptide- chemical complex (i.e. Cor1-C420-cinnamaldehyde and cysteine-2,4- dinitrochlorobenzene) suggesting that the complex formation may be partially reversible. This information suggests that data generated by the DPRA in high-throughput format involving the screening of hundreds of chemicals simultaneously, may not be accurate. Poor aqueous solubility of ERS in in vitro assays is a considerable challenge. To address this issue, the solubility of five ERS using a range of solvent:reaction buffer combinations was compared. In brief, a solvent comprising a 1:1 methanol:acetonitrile containing 1% tert-butanol was effective in solubilising these five ERS in reaction buffer. Using this optimised solvent system for dissolution of DGEBA, TMPTGE, THETGE and PPGE, the DPRA data generated were significantly correlated with the LLNA data on skin sensitisation with the exception that PEGGE was positive in the DPRA but classified as a non-sensitiser in the LLNA. In summary, my findings show that there are many challenges to be overcome in future work beyond the scope of my PhD research project in terms of adapting the DPRA and h- CLAT assays to high-throughput format in order to provide accurate information on the skin sensitisation potential of novel industrial chemicals. ii Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. iii Publications during candidature Peer-reviewed journal articles 1. Wong C.L., Ghassabian S, Smith M.T. and Lam A (2015). In vitro methods for hazard assessment of industrial chemicals – opportunities and challenges. Frontiers in Pharmacology 6:94. doi: 10.3389/fphar.2015.00094. 2. Wong C.L., Lam A, Smith M.T. and Ghassabian S (2015). Optimization of the performance of the direct peptide reactivity assay (DPRA) for assessment of the skin sensitization potential of chemicals. Frontiers in Pharmacology (under review). Conference Abstracts 1. Wong, C.L., Lam, A.L., Wyse, B.D., Smith, M.T. In vitro assessment of chemical sensitisation potential using the human cell line activation test (h-CLAT). In: Abstracts of the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists (ASCEPT) 46th Annual Scientific Meeting; 2012 Dec 2-5, Sydney, Australia. Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists. (Poster) Awarded a travel grant by ASCEPT to present in the 46th Annual Scientific Meeting Neville-Percy prize finalist 2. Wong, C.L., Lam, A.L., Ghassabian, S., Smith, M.T. Evaluation of in vitro assays as alternative strategies in predicting skin sensitisers. In: Abstracts of the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists (ASCEPT) 47th Annual Scientific Meeting; 2013 Dec 1-4, Melbourne, Australia. Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists. (Poster) Awarded a travel grant by ASCEPT to present in the 47th Annual Scientific Meeting iv 3. Wong, C.L., Lam, A.L., Ghassabian, S., Smith, M.T. Evaluation of the direct peptide reactivity assay (DPRA) in identifying epoxy resins with skin sensitizing potential. In: Abstract of the Alternatives to Animal Experimentation (ALTEX) 9th World Congress on Alternatives and Animal Use in the Life Sciences (WC9); 2014 Aug 24-28; Prague, Czech Republic. (Poster) Awarded travel grants by Alternatives Congress Trust (ACT) Germany, Stiftung zur Förderung von Ersatz- und Ergänzungsmethoden zur Einschränkung von Tierversuchen (SET) Foundation (Germany) and Info Kinetics Sdn. Bhd. (Malaysia) 4. Wong, C.L., Lam, A.L., Ghassabian, S., Smith, M.T. Evaluation of the direct peptide reactivity assay (DPRA) in identifying epoxy resins with skin sensitizing potential. In: Abstract of the Translation Research Excellence (TRX14); 2014 Oct 24; Brisbane, Australia. (Poster) 5. Wong, C.L., Lam, A.L., Ghassabian, S., Smith, M.T. Evaluation of the direct peptide reactivity assay (DPRA) in identifying epoxy resins with skin sensitizing potential. In: School of Pharmacy Research Higher Degree Day; 2015 Feb 18; Brisbane, Australia. (Poster) v Publications included in this thesis 1. Wong C.L., Ghassabian S, Smith M.T. and Lam A (2015). In vitro methods for hazard assessment of industrial chemicals – opportunities and challenges. Frontiers in Pharmacology 6:94. doi: 10.3389/fphar.2015.00094 - incorporated as Chapter 1 (refer to Appendix for published paper) Contributor Statement of contribution Author Wrote (70%) and edited the paper (60%) Chin Lin Wong (Candidate) Author Edited paper (5%) Sussan Ghassabian Author Edited paper (15%) Maree T.