Basal, MMS and 3R4F CS induced DNA damage in left and right lung lobes

Prior to initiation of the inhalation study, additional method development was performed to determine whether the right and left lung lobes had comparable basal, MMS induced and 3R4F CS induced DNA damage. To measure MMS induced DNA damage in vitro, Sprague-Dawley rats (8 females, 9–11 weeks, Charles River Laboratories) were acclimatized for 2 days, and AEC II were isolated from the left and right lobes as detailed above. Isolated AEC II were incubated with PBS or 750 μM MMS for 1 h at 37 °C, and the level of DNA damage was identified by the Alkaline Comet assay (Dalrymple et al., 2015). To measure 3R4F CS induced DNA damage in lung left and right lobes, Sprague-Dawley rats (12 females, 9–11 weeks, Charles River Laboratories) were acclimatized for 8 days in accordance with OECD 413 (OECD, 2009). Animals (six per group) were subsequently exposed for 5 days to sham air or 800 μg/L of 3R4F CS WTPM for 1 h/day, as described above. AEC II were isolated from right and left lobes, and the level of DNA damage was identified by the Alkaline and Modified Alkaline Comet assays as described in Dalrymple et al. (2015).