Mold was nominated to the NTP for toxicological characterization and based on their inhalation, mycological, and immunological expertise, NIOSH was engaged as a partner to conduct multiple sub-chronic (13-week) exposure studies to individual organisms or mixtures of molds mimicking a real-world exposure. NIOSH has developed a system that allows for precisely controlled exposures to particulates via inhalation. This approach overcomes a number of limitations associated with previous mold exposure studies in experimental animal models that have used less relevant means of exposure to assess fungal toxicity. The acoustical generation system (AGS) developed as part of this partnership will deliver fungal test articles to mice in a manner that simulates natural indoor fungal contaminant exposure. Aspergillus fumigatus was selected as a model organism for the initial studies. Microscopic and aerodynamic analysis of the aerosolized A. fumigatus indicated that it was a homogenous spore suspension with no detectable ultrastructural change. Deposition studies confirmed that spores were localized to the upper and lower respiratory tract in the exposed mice. A modified local lymph node assay was conducted to evaluate potential sensitization and determine the optimal dose for subsequent studies. Deposition of 1 x 105 spores induced a significant immune response without body weight loss, and was selected as the optimal dose for future inhalation studies. A 13-week subchronic toxicity study was completed in BALB/cJ mice exposed to two different doses of A. fumigatus conidia (104 and105), twice a week for a period of 13 weeks. Exposures were conducted using the AGS and animals inhaled dry aerosols of mono-dispersed A. fumigatus conidia through the nose only chamber. Histopathological analysis revealed dose-dependent pulmonary inflammation, cellular infiltration, goblet cell metaplasia, and peribronchial and perivascular collagen deposition. These responses were accompanied by an increase in total serum IgE and development of A. fumigatus-specific IgG antibodies. NIOSH has also conducted a 13-week subchronic study to examine immune endpoints in B6C3F1/N mice. This study will provide pulmonary and immunologic endpoints that will anchor a subsequent toxicology study. In addition, it will allow for a comparison of immunologic changes between animals predisposed to allergy (Balb/cJ) and those with an intermediate phenotype (B6C3F1/N). Preliminary results suggest that repeated subchronic exposures to viable A. fumigatus conidia lead to an allergic phenotype even in the B6C3F1/N mouse. This response was not observed in mice treated with heat-inactivated conidia. The results of these studies further support that fungal viability is a critical component in the induction of allergy and toxicity. A 13-week subchronic study that will focus on the characterization of toxicological endpoints and aim to produce an exposure scenario that reflects human exposure conditions found in damp or water-damaged buildings was begun in August of 2014.
