INTRODUCTION

Carcinogenic chemicals can be broadly classified based on their mode of action, with a major focus on genotoxic and non-genotoxic mechanisms. Genotoxic carcinogens directly interact with the genetic material of cells, causing mutations, chromosomal fragmentation, or rearrangements. These alterations can disrupt normal cellular functions, leading to uncontrolled cell proliferation and ultimately cancer. Genotoxicity of chemicals can be evaluated in various in vitro and in vivo assays, which are mainly designed to evaluate mutagenicity potential, chromosomal damage, and DNA damage /repair pathways interruption. Due to the recent restrictions in the use ofin vivo genotoxicity assays, there is a need for biologically relevant new approach methodologies (NAMs) to be used as animal alternatives for evaluating genotoxic potential of chemicals that hadin vitro positive results. The Chicken and related Turkey Egg Genotoxicity Assays (CEGA and TEGA, respectively) (Williams et al., 2014; Iatropoulos et al., 2017; Kobets et al., 2018b; Kobets et al., 2016; 2018a), were developed as metabolically competent (Kobets et al., 2018b; Perrone et al., 2004) NAMs for genotoxicity screening to potentially replace short-termin vivo studies required for human safety assessment. CEGA uses fertilized, specific pathogen free eggs from the white leghorn chicken of undetermined sex. Since the termination of the embryos in CEGA is conducted on incubation day 11, at least 10 days before hatching, discomfort to the organism is precluded, as the nervous system of the embryos is not completely developed (Hughes 1953). Thus, in compliance with Animals (Scientific Procedures) Act 1986, CEGA is not considered to be an animal model. CEGA evaluates two different endpoints, DNA adducts by the means of the NPL assay (Phillips and Arlt, 2014; Randerath et al., 1981; Reddy and Randerath, 1986) and DNA strand breaks using the alkaline single cell gel electrophoresis (comet) assay (Brendler-Schwaab et al., 2005; OECD, 2016; Tice et al., 2000). These are indicative of DNA damage produced by either direct or indirect mechanisms. Both techniques are widely used for the evaluation of chemical-induced DNA damage (Himmelstein et al., 2009 and also makes it possible to elucidate the mode of action of chemical carcinogens. Additionally, fetal avian livers express majority of the phase-I and phase-II biotransformation enzymes which can detect chemicals inducing DNA damage post metabolic transformation (Kobets et al., 2018b; Perrone et al., 2004; Rifkind et al., 1979) and they can also efficiently mimic detoxification of chemicals similar to rodent models. Genotoxicity can be induced by direct DNA activity of the parent chemical and/or its metabolite. As such, metabolism plays a crucial role in the bioactivation of many chemicals. This process is often required for the formation of reactive electrophilic intermediates that can then directly react with DNA (Kobets et al., 2019). Bioactivation of different classes of chemicals may differ and produced metabolites may interact with different sites on macromolecules including DNA. Since many in vitro systems lack an intrinsic ability to metabolize chemicals, the induced rat liver S9 fraction is used as an exogenous metabolic activation system (Ames et al, 1973; Paolini et al, 1997). However, this exogenous source of metabolic enzymes does not include those that are important for phase II detoxification. Hence, current in vitro testing systems generate high number of misleading outcomes in testing and prediction of carcinogens (Kirkland et al., 2007). For analysis of the chicken egg liver response to a xenobiotic, a polycyclic aromatic hydrocarbon, benzo[a]pyrene (B(a)P) was chosen. Many of the chemicals that belong to this group are genotoxic carcinogens (Urwin et al., 2024). Carcinogenic activity of B(a)P involves activation of the Aryl hydrocarbon receptor (AhR), which in turn binds to AhR nuclear translocator and induces the expression of genes involved in B(a)P bioactivation and detoxification. These genes are the cytochrome P450 (CYP) genes CYP1A1, CYP1A2, CYP1B1, as well as glutathione transferase (GST) and Uridine diphosphate (UDP)-glucuronosyltransferase (UGT-1). In order to exhibit genotoxicity, B(a)P requires oxidation by phase I CYP1A1 into B(a)P-7,8-epoxide, which through hydration by microsomal epoxide hydrolase is metabolized to B(a)P-7,8-dihydrodiol (BPD) (Figure 1). BPD is then metabolized to B(a)P-7,8-dihydrodiol-9,10-epoxide (BPDE) by second CYP reaction (Kim et al., 2005). BPDE contains an epoxide ring which is highly reactive with DNA in a time dependent manner. In vitro , B(a)P consistently produced negative outcomes in mutagenicity and clastogenicity studies in the absence of metabolic activation, only demonstrating positive outcomes in the presence of exogenous S9 fraction (EPA, 2017).