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The purpose of this study was to introduce an experimental radiobiological setup used for  in vivo  irradiation of a mouse leg target in multiple positions along a proton beam path to investigate normal tissue- and tumor models with varying linear energy transfer (LET). We describe the dosimetric characterizations and an acute- and late-effect assay for normal tissue damage. The experimental setup consists of a water phantom that allows the right hind leg of three to five mice to be irradiated at the same time. Absolute dosimetry using a thimble (Semiflex) and a plane parallel (Advanced Markus) ionization chamber and Monte Carlo simulations using Geant4 and SHIELD-HIT12A were applied for dosimetric validation of positioning along the spread-out Bragg peak (SOBP) and at the distal edge and dose fall-off. The mice were irradiated in the center of the SOBP delivered by a pencil beam scanning system. The SOBP was 2.8 cm wide, centered at 6.9 cm depth, with planned physical single doses from 22 to 46 Gy. The biological endpoint was acute skin damage and radiation-induced late damage (RILD) assessed in the mouse leg. The dose–response curves illustrate the percentage of mice exhibiting acute skin damage, and at a later point, RILD as a function of physical doses (Gy). Each dose–response curve represents a specific severity score of each assay, demonstrating a higher ED50 (50% responders) as the score increases. Moreover, the results reveal the reversible nature of acute skin damage as a function of time and the irreversible nature of RILD as time progresses. We want to encourage researchers to report all experimental details of their radiobiological setups, including experimental protocols and model descriptions, to facilitate transparency and reproducibility. Based on this study, more experiments are being performed to explore all possibilities this radiobiological experimental setup permits.

An experimental setup for proton irradiation of a murine leg model for radiobiological studies