Developmental toxicity screening of methylene blue in a chick embryo in ovo model

Methylene blue (MB) is a clinically important agent used for acquired methemoglobinemia and selected critical care indications, yet its effects during early embryogenesis remain poorly defined. This study evaluated developmental toxicity following in ovo MB exposure, characterized dose-dependent embryolethality, estimated the median lethal dose (LD50), and assessed developmental outcomes across a lower-dose range.An initial analytical cohort of 360 fertilized chicken eggs was allocated to two experimental phases. In the lethal-dose phase, MB was administered into the air chamber at doses 1, 2, 4, or 8 mg/kg. In the low-dose phase, MB was administered at doses of 0.067, 0.125, 0.250, or 0.500 mg/kg. Each phase included a sham-control group and a saline vehicle-control group. Embryonic mortality, developmental progression, relative embryo weight, crown-rump length, and hatch-related outcomes were recorded during incubation. Mortality data were analyzed using control-corrected probit regression. In the lethal-dose phase, mortality increased dose-dependently, reaching 23.3%, 43.3%, 90.0%, and 100.0% at 1, 2, 4, and 8 mg/kg, respectively. The estimated LD50 was 2.01 mg/kg, with a 95% confidence interval of 1.68-2.40 mg/kg. In the low-dose phase, MB concentrations from 0.067 to 0.500 mg/kg did not significantly affect mortality, relative embryo weight, or crown-rump length compared with controls. These findings indicate a steep lethal response to MB in this model, while 0.500 mg/kg may be regarded as a provisional no-observed-adverse-effect level (NOAEL) candidate under the present experimental conditions. Direct clinical extrapolation is limited because the in ovo model lacks maternal metabolism, placental transfer, and fetal clearance.