The purpose of the present study was to explore acute and

sub-chronic effects of airway exposure to biopesticides, with TPCA-1 cell line focus on airway irritation, lung inflammation and clearance of Bt from the lungs. Initially, dose-response and time-response studies were conducted using i.t. instillations. To simulate occupational exposures, mice were in a subsequent experiment exposed repeatedly by inhalation to aerosolised commercially formulated biopesticides based on Bt israelensis or Bt kurstaki. Methods Animals The exposures were performed on BALB/cJ female mice (Taconic M&B, Ry, Denmark), 6-8 weeks old, body weight 18-22 g. Animals were housed up to 10 animals per Small molecule library cage (425

× 266 × 150 mm) and drinking water and food (Altromin no 1324 Brogaard Denmark) was provided ad libitum. Light/dark cycles were at 12 hours and room temperature and relative humidity was kept at 19-22°C and 40-60%, respectively. All protocols were approved by the Danish Animal Experiments Inspectorate. Bacterial suspensions and CFU determinations The bacterial suspensions were prepared from commercially available insecticides Vectobac® (Bt israelensis) and Dipel® (Bt kurstaki) from Valent Biosciences (Selleckchem Sapanisertib Sumitomo Chemical Agro Europe, Lyon, France). The suspensions for aerosol generation and intratracheal instillation were prepared from the formulated products by suspending them

in sterile, endotoxin-free water. To reduce viscosity (caused by additives) during the high dose instillations of Dipel®, mice in one group (experiment 4, cf. Table 1) received product that was subjected to a washing procedure: the Dipel® was suspended and centrifuged and supernatant discharged. This procedure was repeated twice. The final precipitate was re-suspended in sterile water and adjusted for CFU counts. Table 1 Experimental overview Exp.No. Aim of experiment Number of mice Exposure method Substance Time Endpoint Endpoint Corresponding figure 1 Validation of Inhalation dose GNA12 10 Inhalation (1 hour) Vectobac® 1 h CFU from total lung homogenate Figure 1 2 Validation of CFU recovery from BALF 8 Inhalation Vectobac® 1 h CFU from BALF and lavaged lungtissue None 3 Dose- response relationship B.t israelensis 25 Instillation Vectobac® 24 h Inflammatory cells in BALF Figure 2 4 Time- response relationship B.t israelensis or B.t kurstaki 42 Instillation Vectobac® or Dipel® (washed) 4 h, 24 h, 4 days CFU and inflammatory cells in BALF Figure 3 5 Sub-chronic effects of i.t instillations of B.t israelensis or B.t kurstaki 20 Instillation Vectobac® or Dipel® 70 days CFU, Inflammatory cells in BALF, Histology Figure 4 Figure 5 6 Sub-chronic effects of repeated inhalations of B.t israelensis or B.