Until today, there is not any official registry of the different routes of systemic ozone therapy. With the aim to prove the safety of this therapy, several ways of ozone (O3) applications were evaluated, such as rectal insufflation (RI), major autohemotherapy (M-AHT) and intraperitoneal application (IP). For RI, some toxicological tests were performed such as: acute, sub-chronic, mutagenic (bone marrow chromosomic aberrations and micronucleus), teratogenic  and irritation studies (at 40 mg/L and 10 mL during 15 days). For M-AHT, acute toxicological (in rats with 21, 47 and 64 mg/L and a volume of 2 mL with daily evaluation up to 15 days) and mutagenic (chromosomal aberration analysis, micronucleus assay and sister chromatid exchange test) studies were performed. For IP application, subchronic (O3 was administered during 15 days in mice at concentrations of 11, 29 and 35 mg/L and volumes of 80 mL/kg) and genotoxic (cytotoxic and clastogenic activity) studies were evaluated. All the toxicological studies performed for the O3 RI fulfilled the requirements established for an official registry (no deleterious effects were observed). However, more toxicological studies must be done with respect to M-AHT and IP O3 applications in order to prove the complete safety of these ways.