Efficacy and safety of free and nanodrugs on the chicken embryo model via different routes

Background and purpose: The chicken embryo is increasingly used as an alternative preclinical model for pharmacokinetic and toxicity studies; however, standardized protocols considering different inoculation routes are still lacking. This study aimed to evaluate how inoculation routes influence toxicity and pharmacokinetics in the chicken embryo model, using free and nanoencapsulated norfloxacin, indomethacin, and lidocaine as model compounds. Experimental approach: On day 10 of development, embryos received each formulation via four routes: shell membrane, allantois, chorioallantoic membrane, or amnion. Toxicity was assessed after six days based on mortality, embryo weight, and histological alterations in the liver and kidney. Pharmacokinetic parameters of commercial and nanoformulated norfloxacin were determined in serum and allantoic fluid following a single dose administered through the same routes. Key results: Norfloxacin did not affect embryo weight, whereas commercial lidocaine increased it, and both free and nanoencapsulated indomethacin significantly reduced it. Histological lesions were observed exclusively after amniotic administration of nanoformulations, including renal inflammation or degeneration and hepatic degeneration, depending on the compound. Norfloxacin reached maximum serum concentration 1 h post-administration for all routes except the amnion, which showed a delayed peak at 24 h. Norfloxacin was detected in allantoic fluid only after amniotic inoculation. Conclusions and implications: Chicken embryos are suitable for pharmacokinetic and toxicity assessments, but experimental outcomes are strongly influenced by inoculation route and formulation properties, underscoring the need for route-specific standardization.