A tiered testing strategy has been developed to evaluate the potential of tobacco processes, ingredients, or technological developments to change the biological activity resulting from burning tobacco. The strategy is based on comparative chemical and biological testing. Expanded shredded tobacco stems (ESS) constitute an example of a common tobacco components expansion process currently used in the manufacture of cigarettes to increase the tobacco blend filling capacity. As part of the toxicological evaluation of ESS, test cigarettes containing 9.5%, 18.5%, and 25% ESS were compared to control cigarettes containing 0% ESS. Testing included mainstream cigarette smoke chemistry studies, genotoxicity studies (Ames and sister chromatid exchange), a 13-week inhalation study in Sprague-Dawley rats, and a 30-week dermal tumor promotion study in SENCAR mice. Collectively, data indicated that cigarettes with and without ESS had a similar toxicological profile in this test battery.