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

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

Anne North  1 , Katherine Ling  2 , Guillaume Ricaud  3 , Leon F Stankowski Jr  4 , Jennifer A Daly  5 , Stanley Bentow  2 , Laurence Corash  2 , Richard J Benjamin  2 , Nina Mufti  2
Affiliations

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

Anne North et al. Transfusion. 2024 Jun.

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.

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

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References

REFERENCES

    1. Aydinok Y, Piga A, Origa R, Mufti N, Erickson A, North A, et al. Amustaline‐glutathione pathogen‐reduced red blood cell concentrates for transfusion‐dependent thalassaemia. Br J Haematol. 2019;186(4):625–636.
    1. Brixner V, Kiessling AH, Madlener K, Muller MM, Leibacher J, Dombos S, et al. Red blood cells treated with the amustaline (S‐303) pathogen reduction system: a transfusion study in cardiac surgery. Transfusion. 2018;58(4):905–916.
    1. Snyder EL, Sekela ME, Welsby IJ, Toyoda Y, Alsammak M, Sodha NR, et al. Evaluation of the efficacy and safety of amustaline/glutathione pathogen‐reduced RBCs in complex cardiac surgery: the red cell pathogen inactivation (ReCePI) study‐protocol for a phase 3, randomized, controlled trial. Trials. 2023;24(1):799.
    1. North A, Ciaravino V, Mufti N, Corash L. Preclinical pharmacokinetic and toxicology assessment of red blood cells prepared with S‐303 pathogen inactivation treatment. Transfusion. 2011;51(10):2208–2218.
    1. North AK, Mufti N, Sullivan T, Corash L. Preclinical safety assessment of pathogen reduced red blood cells treated with amustaline and glutathione. Transfusion. 2020;60(2):358–366.

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