The Chicken Egg Genotoxicity Assay (CEGA) is an avian egg-based model that utilizes the livers of developing chicken embryo-fetuses to assess the ability of chemicals to produce direct DNA damage. The main goal of the study was to evaluate target tissue exposure and metabolism in the CEGA to assess its suitability as a biologically relevant new approach methodology (NAM) for detecting genotoxic potential of chemicals. An imaging study using two-photon excitation microscopy following administration of a fluorescent dye (acridine orange) verified that chemicals following administration into the air sac of the fertilized chicken egg reach the target organ, liver. Additionally, a metabolism study using liquid chromatography with high resolution mass spectrometry (LC/MS), conducted after administration of benzo[a]pyrene (B(a)P) according to the CEGA protocol, confirmed the formation of sufficient amounts of reactive metabolite(s) responsible for genotoxic effects of a parent compound upon reaching the target tissue. Moreover, RNA sequencing study revealed that B(a)P in embryo-fetal chicken livers significantly upregulated several genes responsible for the activity of CYP1A1 enzyme which is critical for bioactivation of B(a)P. These findings support previous reports in CEGA, where B(a)P produced DNA damage in the liver tissues in the form of strand breaks and adducts. Overall, the findings in the study support the conclusion that the CEGA can be considered a robust potential alternative to animal testing strategy for assessing the genotoxic potential of chemicals