Dermal Absorption of Nanomaterials Titanium Dioxide and Zinc Oxide Based Sunscreen Role of Size and Surface Coating Environmental project No. 1736, 2015 Title: Editing: Dermal Absorption of Nanomaterials Titanium Christiane Beer, Department of Public Health, Aarhus University, Dioxide and Zinc Oxide Based Sunscreen Denmark Karin Stenderup, Department of Dermatology, Aarhus University Hospital, Denmark Jing Wang, iNANO Center, Aarhus University, Denmark Duncan S. Sutherland, iNANO Center, Aarhus University, Denmark Jens R. Nyengaard, Department of Clinical Medicine, Aarhus University , Denmark Published by: The Danish Environmental Protection Agency Strandgade 29 1401 Copenhagen K Denmark www.mst.dk/english Year: ISBN no. 2015 978-87-93352-53-7 Disclaimer: When the occasion arises, the Danish Environmental Protection Agency will publish reports and papers concerning research and development projects within the environmental sector, financed by study grants provided by the Danish Environmental Protection Agency. It should be noted that such publications do not necessarily reflect the position or opinion of the Danish Environmental Protection Agency. However, publication does indicate that, in the opinion of the Danish Environmental Protection Agency, the content represents an important contribution to the debate surrounding Danish environmental policy. Sources must be acknowledged. 2 Dermal Absorption of Nanomaterials Titanium Dioxide and Zinc Oxide Based Sunscreen Contents Foreword .................................................................................................................. 5 Executive Summary ................................................................................................... 6 Resumé .................................................................................................................... 11 1. Introduction ..................................................................................................... 16 1.1 Scientific background ........................................................................................................... 16 1.2 Objective of the project ......................................................................................................... 17 1.3 Experimental methods ......................................................................................................... 18 1.4 Structure of the report .......................................................................................................... 18 2. Material and Methods....................................................................................... 19 2.1 Overview of the experimental setup ..................................................................................... 19 2.2 Nanoparticles ........................................................................................................................ 19 2.3 Sunscreen formulation ......................................................................................................... 21 2.4 Preparation of nanoparticle containing sunscreen ............................................................. 21 2.5 Nanoparticle characterization ............................................................................................. 23 2.5.1 TEM and EDX analysis of nanoparticle dry powders .......................................... 23 2.5.2 Dynamic light scattering (DLS) analysis of the nanoparticle size and surface charge in MilliQ water .............................................................................. 23 2.5.3 In situ observation of Nanoparticles in sunscreen using an optical microscope ............................................................................................................ 23 2.5.4 Acoustic attenuation spectroscopy (AAS) ............................................................ 24 2.6 In vitro and in vivo skin models ......................................................................................... 24 2.6.1 Treated surface area .............................................................................................. 24 2.6.2 Time of exposure ................................................................................................... 24 2.6.3 Dosage of nanoparticles ........................................................................................ 24 2.6.4 In vitro EpiDerm™ system (MatTek) – normal, human 3D epidermis model ..................................................................................................................... 26 2.6.5 In vivo mouse model for acute irritant contact dermatitis (ICD) ........................ 31 2.6.6 In vivo xenograft human skin model ................................................................... 32 2.6.7 Statistical analysis ..................................................................................................37 3. Results ............................................................................................................. 38 3.1 Nanoparticle preparation and characterization ................................................................. 38 3.1.1 Characterization of the pre-dispersant ................................................................ 38 3.1.2 Characterization of sunscreen .............................................................................. 38 3.1.3 Characterization of the nanoparticle powders ..................................................... 39 3.1.4 Particle size distribution of nanoparticles dispersed in MilliQ water, pre- dispersant C12-C15 alkyl benzoate and sunscreen .............................................. 42 3.1.5 Acoustic attenuation spectroscopy (AAS) measurement of the particle size distribution in sunscreen ............................................................................... 46 3.1.6 Conclusion nanoparticle characterization ........................................................... 48 3.2 Dermal absorption/penetration of TiO2 and ZnO NPs in in vitro EpiDerm™ skin model .................................................................................................................................... 48 3.2.1 Effect of sunscreen on skin model permeability – TEER measurements .......... 48 Dermal Absorption of Nanomaterials Titanium Dioxide and Zinc Oxide Based Sunscreen 3 3.2.2 Investigation of the transport of TiO2 and ZnO NPs through EpiDermTM skin models using ICP-MS .................................................................................... 49 3.2.3 Cell viability ........................................................................................................... 52 3.2.4 Cytokine analysis ................................................................................................... 53 3.2.5 Histology and transmission electron microscopy ................................................ 58 3.2.6 Conclusion in vitro EpiDermTM skin model......................................................... 60 3.3 In vivo skin models ............................................................................................................... 61 3.3.1 In vivo TPA irritant contact dermatitis (ICD) mouse model ............................... 61 3.3.2 Xenograft human skin model ............................................................................... 69 3.3.3 Conclusion in vivo skin models ............................................................................ 72 4. Discussion ........................................................................................................ 73 5. Conclusion ....................................................................................................... 79 References .............................................................................................................. 80 Appendix 1: Nanoparticle information as provided by the manufacturer ........... 85 Appendix 2: Transmission electron microscopy ................................................ 89 Appendix 3: Weight and ear thickness in in vivo mouse model for acute irritant contact dermatitis after exposure to sunscreen with or without nanoparticles ........................................................................... 105 Appendix 4: Weight measurements in in vivo human xenograft skin model ..... 112 Appendix 5: ICP-MS measurement data of medium below in vitro EpiDerm skin models and TEER of the tissues after 20h of exposure ..................... 114 Appendix 6: Cytokine release of in vitro EpiDerm skin models after 20h exposure (AE) and 3 days after ended exposure ...................................... 117 Appendix 7: Test report ICP-MS measurements of the in vitro and in vivo skin models: cell culture medium, mice ears and human skin ................. 131 Appendix 8: Certificate of analysis for the used lots of EpiDerm™ skin model ................................................................................................... 136 4 Dermal Absorption of Nanomaterials Titanium Dioxide and Zinc Oxide Based Sunscreen Foreword In 2011 the Danish government and the Red-Green Alliance (a.k.a. Enhedslisten) decided on an initiative called “Bedre styr på nano” (“Better control of nano”) for the Danish Finance Act of 2012. This initiative is running for four years (2012-2015) and includes several projects for the use of nanomaterials in products on the Danish market and their consequences on consumers and the environment. Furthermore, the aim is to clarify possible risks that might be associated