# The Chicken Egg Genotoxicity Assay (CEGA): Assessing Target Tissue Exposure and Metabolism in the Embryo‐Fetal Chicken Livers

## TL;DR

The Chicken Egg Genotoxicity Assay (CEGA) is a new method to test if chemicals cause DNA damage in chicken embryos' livers, offering a potential alternative to animal testing.

## Contribution

The study demonstrates that CEGA can effectively detect genotoxicity through target tissue exposure and metabolism in avian embryos.

## Key findings

- Chemicals administered into the air sac of fertilized chicken eggs reach the liver, as shown using acridine orange and two-photon excitation microscopy.
- Benzo(a)pyrene is metabolized into reactive intermediates in the embryo-fetal liver, confirmed by LC/MS analysis.
- Benzo(a)pyrene upregulates CYP1A1-related genes in the liver, indicating bioactivation and genotoxic potential.

## Abstract

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 the genotoxic potential of chemicals. An imaging study using two‐photon excitation microscopy after the 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. A metabolism study using liquid chromatography with high resolution mass spectrometry (LC/MS), conducted after the 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 the genotoxic effects of a parent compound upon reaching the target tissue. Moreover, an RNA sequencing study revealed that B(a)P in embryo‐fetal chicken livers significantly upregulated several genes responsible for the activity of the CYP1A1 enzyme, which is critical for the bioactivation of B(a)P. These findings, along with the previously reported DNA damage (i.e., DNA adducts and single‐strand breaks) produced by B(a)P in CEGA, support sufficient target tissue exposure to B(a)P and the ability of avian fetal livers to bioactivate B(a)P to a reactive intermediate. Overall, the findings in the study support the conclusion that the CEGA can be considered a robust potential alternative to the animal testing strategy for assessing the genotoxic potential of chemicals.