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The widespread use of titanium dioxide (TiO 2 ) has raised concerns about potential health risks associated with its cytotoxicity in the cardiovascular system. To evaluate the cytotoxicity of TiO 2 particles, the H9c2 rat cardiomyoblasts were used as a biological model, and their toxicological susceptibility to TiO 2 -anatase and TiO 2 -rutile particles was studied in vitro. The study examined dose and time exposure responses. The cell viability was evaluated based on metabolic inhibition and membrane integrity loss. The results revealed that both TiO 2 -anatase and TiO 2 -rutile particles induced similar levels of cytotoxicity at the inhibition concentrations IC 25 (1.4−4.4 μg/cm 2 ) and IC 50 (7.2−9.3 μg/cm 2 ). However, at more significant concentrations, TiO 2 -rutile appeared to be more cytotoxic than TiO 2 -anatase at 24 h. The study found that the TiO 2 particles induced apoptosis events, but necrosis was not observed at any of the concentrations of particles used. The study considered the effects of microstructural properties, crystalline phase, and particle size in determining the capability of TiO 2 particles to induce cytotoxicity in H9c2 cardiomyoblasts. The microstress in TiO 2 particles was assessed using powder X-ray diffraction through Williamson−Hall and Warren−Averbach analysis. The analysis estimated the apparent crystallite domain and microstrain of TiO 2 -anatase to be 29 nm (ε = 1.03%) and TiO 2 -rutile to be 21 nm (ε = 0.53%), respectively. Raman spectroscopy, N 2 adsorption isotherms, and dynamic light scattering were used to identify the presence of pure crystalline phases (>99.9%), comparative surface areas (10 m 2 /g), and ζ-potential values (−24 mV). The difference in the properties of TiO 2 particles made it difficult to attribute the cytotoxicity solely to one variable.