The in vivo Comet assay can evaluate the genotoxic potential of a chemical in theoretically any tissue that can be processed to a single cell suspension. This flexibility enables evaluation of point-of-contact tissues using a relevant route of test material administration; however, assessing cytotoxicity is essential for the interpretation of Comet results. Histopathological evaluation is routinely utilized to assess cytotoxicity, but temporal- and cell-specific considerations may compromise applicability to the Comet assay. In the present study, 1,1′-methylenebis(4-isocyanatobenzene) (4,4-MDI) was administered to rats for 6 hours by nose-only inhalation and the Comet assay was conducted to evaluate genotoxicity in the site-of-contact tissue (bronchoalveolar lavage cells) and distal tissues (liver and glandular stomach). Given the reactive nature of MDI, cellular and molecular metrics at the site-of-contact, including inflammation, macrophage activation, apoptosis/necrosis, and oxidative stress, in addition to the standard systemic measures of toxicity were used to set appropriate exposure concentrations. In the range-finding study a concentration of 4 mg/m3 was considered the maximum noninflammatory concentration, hence target concentrations of 2, 5, and 11 mg/m3 were selected for the Comet study. In the lung lavage, MDI exposure substantially increased total protein and β-glucuronidase along with cellular apoptosis. While MDI did not increase the Comet assay response (% tail DNA) in any of the tissues examined, the positive control (EMS) significantly increased % tail DNA in all tissues. In total, these data indicate that appropriate cellular and molecular measurements may facilitate dose selection to discern cellular status in the Comet assay.