The toxicity of a nebulized mixture of nicotine and pyruvic acid, nicotine, and sodium pyruvate, was characterized in a repeated dose inhalation study. Sprague Dawley rats were nose-only exposed for 6 hours per day, 5 days per week in a 28-day study to either filtered air, PBS (solvent control), nicotine (50 µg/l), sodium pyruvate (33.9 µg/l), or three concentrations of the nicotine-pyruvic acid mixture (nicotine concentrations were 18, 25, and 50 µg/l, with equimolar concentrations of pyruvic acid) . The test aerosols were produced using jet collision nebulizers and were in the respirable particle size range. Compared to the air control, the PBS and pyruvate groups showed no treatment-related effects. Effects in the rats exposed to the test atmospheres containing nicotine were as follows: In life observation showed a reduced weight gain. However, food consumption was not significantly reduced over the study duration. No exposure-related mortality or abnormal gross pathological findings were observed. Organ weight changes were noted in liver (increase) and spleen (decrease). Clinical pathology evaluation showed slightly higher erythrocyte and haemoglobin values in nicotine-exposed females relative to control. In addition the neutrophil count was higher whereas the lymphocyte count was lower in all animals exposed to nicotine. For the clinical chemistry, blood alkaline phosphatase and alanine aminotransferase activities were higher; cholesterol and glucose concentrations were lower than in the control rats. Histopathological findings in the non-respiratory tract consisted of an the appearance of vacuolization in the liver. In the respiratory tract, mild laryngeal epithelial hyperplasia and squamous metaplasia was observed, as well as an increased epithelial thickness. A low level of lung inflammation was found in the nicotine-exposed rats. Most of the effects were nicotine exposure concentration-dependent.In conclusion, minor biological effects related to inhalation of nicotine either alone or in combination with pyruvic acid were observed in a 28-day inhalation study. The nicotine-related effects were not modulated by pyruvic acid.