Objective: Before each clinical trial of advanced therapy medicinalproducts (ATMPs), preclinical s tudies on animal modelsof diseases are required to characterize the mechanisms ofaction and evaluation the toxicity, the dosage, and the route ofadminis tration of the new medicine. The aim of this s tudy wasto inves tigate the sys temic toxicity of two skin subs titutes, RoyinSheetand RoyinGraf, in both subacute and subchronic phasesof wounds in rat. RoyinSheet, a cryopreserved cultured epidermis(human keratinocyte sheet), and RoyinGraf, a bilayered livingcellular cons truct from human fibroblas t and keratinocytewere developed in the ATMP-TDC center at Royan Ins titute.Materials and Methods: The potential sys temic effects of twoskin subs titutes were s tudied after transplantation of RoyinSheetin burn wounds and RoyinGraf in full-thickness wounds in rats.In each subacute and subchronic toxicity s tudy, the male andfemale Wis tar rats were randomly assigned into three groups(treatment, sham, and control). The behavior, body weight,and food- and water intake were assessed during these phases.Two weeks (for subacute phase) and ۱۳ weeks (for subchronicphase) pos t-transplantation, the animals were sacrificed andurine and blood samples and also major organs (liver, kidneys,heart, lung, Bone marrow (BM)) were harves ted from each rat.All tissues were processed and s tained with hematoxylin andeosin (H&E) and BM was s tained with Giemsa for light microscopicexamination.Results: During the s tudy, death was observed in four animalswithin ۲۴ hours pos t-surgery in the sham group, maybe dueto the anes thesia. No animal death occurred in the treatmentgroups. Based on the two-way ANOVA s tatis tical analysis, nosignificant differences were observed in parameters of bodyweight, water, and food intake in both subacute and subchronicphases. Urine analysis did not show pathologic effects in treatmentversus sham and control groups. Hematological, biochemical,and blood coagulation tes ts indicated that the triglycerideparameter in the subacute phase of RoyinSheet product andplatelet-, glucose- and urea parameters in the subacute phase ofRoyinGraf product were significantly different between shamand treatment groups in comparison to healthy controls (onewayANOVA s tatis tical analysis, P value <۰.۰۵). However,there was not any significant difference between sham and treatmentgroups. Also, no pathologic abnormities were seen in thes tructure of vital organs (liver, lung, heart, and kidney) and BMin both subacute and subchronic phases. No signs of his tologicalchanges such as inflammation, abnormal tissues, or necrosiswere observed in the heart, lung, kidney, liver, and BM in eachgroup.Conclusion: Overall, the results sugges t that local transplantationof skin subs titute RoyinSheet and RoyinGraf in both sexesof Wis tar rats do not cause toxicity during the subacute andsubchronic periods of time.