Background <p>Recombinant L-IFN Adenovirus (YSCH-01) is a modified oncolytic adenovirus, which showed good curative effects in a variety of solid tumors. The safety of YSCH-01 needs to be evaluated for non-clinical research.</p> Methods <p>This toxicity and distribution study of YSCH-01 injection via ITU/IV/IP injection in Syrian Hamster was conducted to prepare an investigational new drug (IND) application. The endpoints included mortality/moribundity, clinical observation, body weight, palpable mass examination, food consumption, ophthalmology, and clinical pathology. Toxicokinetics, ADA test, viral shedding, and tissue bio-distribution were analyzed.</p> Results <p>The severely toxic dose in 10% of animals was 1 × 10¹¹ VP/animal, &gt; 2 × 10¹¹ VP/kg, and 6.7 × 10¹¹ VP/kg for ITU groups, IV groups and IP groups, respectively. The antibody titers of recombinant L-IFN adenovirus were significantly increased following YSCH-01 administration, and slightly lower during recovery period. YSCH-01 gDNA can be shed through the administration site, saliva and feces, and almost no urine. In the ITU, IV, and IP injection groups, YSCH-01 was distributed in the whole blood and all collected organs with T_max in most whole blood/tissues of 2.00–4.00&#xa0;days and L-IFN was detected in all tumor and serum samples, with a T_max of 2.00–4.00&#xa0;days, full or partial clearance of YSCH-01 and L-IFN was noted in most tissues/organs or tumor and serum on D28.</p> Conclusions <p>Our study demonstrated the toxicity and potential toxicity of YSCH-01 in Syrian hamsters and evaluated its tissue distribution in hamsters after a single administration. STD10 results in hamsters support the safety of the estimated dosage for future clinical studies.</p> Graphical Abstract <p></p>