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

Transfusion. 2024 Jun;64(6):1097-1108. doi: 10.1111/trf.17854. Epub 2024 May 8.


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.

Keywords: RBC transfusion; hematology–red cells; molecular biology.

MeSH terms

  • Animals
  • CD59 Antigens / genetics
  • Erythrocytes / drug effects
  • Erythrocytes / metabolism
  • Male
  • Membrane Proteins / genetics
  • Mutagenesis / drug effects
  • Mutagenicity Tests* / methods
  • Mutagens / toxicity
  • Rats
  • Rats, Sprague-Dawley*
  • Reticulocytes / drug effects


  • phosphatidylinositol glycan-class A protein
  • CD59 Antigens
  • Membrane Proteins
  • Mutagens

Grants and funding