Establishment of Reference Data for Negative Controls in Alternative Developmental Toxicity Screening Methods Using Chick Embryo-based Models

Abstract

Developmental toxicity studies assess the potential effects of chemical substances and drug candidates on embryonic and fetal development. Mammalian laboratory animals are widely used but are associated with ethical, cost, and animal welfare concerns. In this context, chick embryo-based models have gained attention as alternative tools for developmental toxicity screening; however, the lack of standardized reference data has limited their broader application. This study aimed to establish initial reference data characterizing normal development in chick embryos to support alternative developmental toxicity screening methods. Specific pathogen-free (SPF) fertilized chicken eggs were used under standardized incubation conditions, and four independent experiments were conducted. Mortality rate, morphological developmental parameters (crown-rump length, fore- and hindlimb length, beak length, and third toe length), egg weight, and hatchability rate were recorded and summarized using a total of 132 SPF fertilized chicken eggs. In results, viability decreased from 100% at embryonic day (ED) 0 to approximately 92% by ED 10 and remained stable through ED 20. Morphological parameters increased progressively with development, with crown-rump length reaching 83.01 ± 3.30 mm, forelimb and hindlimb lengths 29.70 ± 1.55 mm and 48.58 ± 3.54 mm, beak length 5.77 ± 0.32 mm and 11.78 ± 0.74 mm, and third toe length 21.62 ± 1.17 mm at ED 20. Egg weight gradually decreased during incubation, embryonic wet weight at ED 20 was 32.54 ± 3.51 g, and the hatchability rate was 78%. This study establishes initial reference data for normal chick embryo development to support early-stage developmental toxicity screening, while highlighting the need for further studies to broaden its applicability to regulatory toxicology.