The purpose of the present study was to explore acute and

sub-chronic effects of airway exposure to biopesticides, with Dinaciclib 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 selleck inhibitor 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 (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 Adenosine triphosphate 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.