In vivo genotoxicity assessment of N‐(‐9 acridinyl)‐b‐alanine hydrochloride (S‐300) using a validated Pig‐a mutagenesis assay

Abstract Background N ‐(‐9 acridinyl)‐b‐alanine hydrochloride (S‐300) is the main byproduct of red blood cell (RBC) amustaline/glutathione(GSH) pathogen reduction, currently undergoing phase III US clinical trials following successful European studies(1–3). Phosphatidylinositol glycan, class A ( Pig‐a ) X‐linked gene mutagenesis is a validated mammalian in vivo mutation assay for genotoxicity, assessed as clonal loss of glycosylphosphatidylinositol‐linked CD59 cell‐surface molecules on reticulocytes (RETs) and RBCs. Methods Male Sprague–Dawley rats received continuous infusion of S‐300 up to the maximum feasible dose (240 mg/kg/day—limited by solubility and volume) for 28 days. Positive controls received a known mutagen by oral gavage on Days 1–3. Plasma levels of S‐300 were assessed by HPLC before, during and after infusion. CD59‐negative RBCs and RETs were enumerated in pre‐dose and Day 28 samples, using a flow cytometric method. Outcome was evaluated by predetermined criteria using concurrent and historical controls. Toxicity was assessed by laboratory measures and necropsy. Results S‐300 reached maximum, dose‐dependent levels (3–15 μmol/L) within 2–8 h that were sustained for 672 h and undetectable 2 h after infusion. Circulating RET levels indicated a lack of hematopoietic toxicity. Necropsy revealed minimal‐mild observations related to poor S‐300 solubility at high concentrations. Pig‐a assessment met the preset acceptability criteria and revealed no increase in mutant RBCs or RETs. Conclusions Maximum feasible S‐300 exposure of rats by continuous infusion for 28 days was not genotoxic as assessed by an Organization for Economic Cooperation and Development‐compliant, mammalian, in vivo Pig‐a gene mutation assay that meets the requirements of International Conference on Harmonization (ICH) S2(R1) and FDA guidances on genotoxicity testing.