The ability to produce direct DNA damage (genotoxicity) underlies the carcinogenic mode of action of various chemicals. As such, genotoxicity endpoints are typically evaluated in a regulatory-approved battery of in vitro tests with potential in vivo follow-up. Growing concern for animal welfare and implementation of new regulations which restrict the use of laboratory animals necessitated the introduction of New Approach Methodologies (NAMs). The avian egg-based (in ovo) models, the Chicken and related Turkey Egg Fetal Liver DNA Damage Assays, were developed as metabolically competent NAMs to potentially replace short-term in vivo genotoxicity assays for chemicals that are genotoxic in vitro. Both models utilize avian fetal livers for the evaluation of endpoints indicative of DNA damage produced by either direct or indirect mechanisms, specifically, the formation of nuclear DNA adducts and strand breaks. Moreover, avian embryos carry genetic and morphologic resemblance to mammals and can be used for an extensive evaluation of other endpoints including histopathology and tissue-specific genomic profiling. Avian fetal livers contain a full complement of metabolizing enzymes and are capable of bioactivation, detoxication, and elimination of xenobiotics. The comprehensive analysis of 87 and 59 chemicals assessed in the chicken and turkey models, respectively, revealed a stronger correlation with the results from in vivo assays demonstrating that in ovo models can detect the genotoxic potential of a broader range of compounds compared to in vitro assays with S9 supplementation. In conclusion, fertilized avian egg fetal liver assays offer a promising alternative to traditional in vivo genotoxicity assays.

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.