Quantitative and toxicological assessment of air-liquid cell exposure to NM-containing spray formulations using a custom-built setup

F.S. Bierkandt, J. Tentschert, H. Jungnickel, P. Laux, A. Luch, P. Bergelt, M. Stintz, E. Visileanu, H. Hinterbichler, H. Steiner, G. Brenn, F. Goni de Cerio, J.D. Brain
German Federal Institute for Risk Assessment (BfR),

Keywords: spray application, aerosolized nanomaterials, cell exposure, quantification


The growing applications of engineered nanomaterials (ENM) and especially their use as constituents in numerous spray products (cosmetics, coating or cleaning products) increase the possibility of human exposure via inhalation. Investigation of such formulations with regard to their deposition behavior and toxic potential is urgently required. Adverse effects associated with aerosol inhalation are influenced by the size and shape of ENM. Amongst others, such as breathing pattern, these factors influence particle where particles deposit in the respiratory system. Moreover, the composition of sprayed formulations impact ENM aerosol toxicity due to the identity and amount of all constituents. These excipients may alter ENM properties like cellular penetration or reactivity. Thus, characterization of spray formulations and their influence on the fate of ENMs in solution as well as after aerosolization are essential and are addressed in this work. The alteration of the ENMs after spray application and their uptake in cellular systems needs to be investigated in toxicological test systems. In order to address the effects of commercially available products, an exposure system producing sprays of realistic formulations, where these formulations can be tested in vitro and in vivo, has been developed. Water and ethanol based formulations containing Ag- and CeO2-ENMs were used to emulate commercial spray products in combination with multiple additives. Since the purpose of such spray application is to water proof textiles, additional surface active substances were added. We selected two perfluorinated silanole additives and examined their influence on ENM toxicity. The particles were selected due to their frequent use based on their bactericidal activity (Ag-ENMs) or their UV stability (CeO2-ENM). Furthermore, they represent two groups of ENMs with different water solubility: Ag-ENMs being more soluble and CeO2-ENMs being rather insoluble and biodurable. To address the importance of particle size, respirable ENM sizes of < 100 nm, ~ 200 nm and micron-sized particles were chosen. A specialized spray chamber design in combination with a commercially available spray can and nozzle was developed and characterized in regard to its aerosolization behavior. This system was coupled online to an air-liquid interface (ALI) exposure chamber exposure of cells and aerosol characterization, utilizing a scanning mobility particle sizer and a condensation particle counter (SMPS, CPC). In addition, a cloud chamber based exposure system was utilized in parallel. Different spray scenarios – varying in spray duration, spray frequency and number – and different ENM formulations were applied during exposure studies of A549 cells. Quantification of silver or of cerium of the aerosol retained by the cells was done using inductively coupled massed spectrometry (ICP-MS). We found that only a fraction of the aerosol entering the ALI exposure system was found in the cells and that this amount depended on the spraying formulation scenario program used. Toxicological assessment of the ENM and additives on the cells was done using viability tests and measurement of inflammatory markers and compared to controls which were only exposed to ENM- or additive-free spray formulations. Our preliminary results indicated that the solvent and additives amount present have major effects.