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

The genotoxic and clastogenic/aneugeneic potentials of four α, ß-unsaturated aldehydes, 2-phenyl-2-butenal, nona-2-trans-6-cis-dienal, 2-methyl-2-pentenal and p-methoxy cinnamaldehyde, which are used as fragrance materials, were assessed in avian fetal livers using the Chicken Egg Genotoxicity Assay (CEGA) and the Hen’s egg micronucleus (HET-MN) assay, respectively. Selection of materials was based on their chemical structures and the results of their assessment in the regulatory in vitro and/or in vivo genotoxicity test battery. Three tested materials, 2-phenyl-2-butenal, nona-2-trans-6-cis-dienal and 2-methyl-2-pentenal, were negative in both, CEGA and HET-MN assays. These findings were congruent with the results of regulatory in vivo genotoxicity assays. In contrast, p-methoxy cinnamaldehyde, which was also negative in the in vivo genotoxicity assays, produced evidence of DNA damage, including DNA strand breaks and DNA adducts in CEGA, however, no increase in the micronucleus formation in blood was reported in the HET-MN study. Pretreatment with a glutathione precursor, N-acetyl cysteine, negated positive outcomes produced by p-methoxy cinnamaldehyde in CEGA, indicating that difference in response observed in the egg and rodent models can be attributed to rapid glutathione depletion. Additionally, the dosing protocols for both HET-MN and CEGA assays are different, which can also be an important contributing factor. Overall, our findings support the conclusion that CEGA and/or HET-MN can be considered as a potential alternative to animal testing as follow-up strategies for assessment of genotoxic potential of fragrance materials with evidence of genotoxicity in vitro.