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

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.

Genotoxicity Assessment of Nanomaterials: Recommendations on Best Practices, Assays, and Methods.

Nanomaterials (NMs) present unique challenges in safety evaluation. An international working group, the Genetic Toxicology Technical Committee of the International Life Sciences Institute's Health and Environmental Sciences Institute, has addressed issues related to the genotoxicity assessment of NMs. A critical review of published data has been followed by recommendations on methods alterations and best practices for the standard genotoxicity assays: bacterial reverse mutation (Ames); in vitro mammalian assays for mutations, chromosomal aberrations, micronucleus induction, or DNA strand breaks (comet); and in vivo assays for genetic damage (micronucleus, comet and transgenic mutation assays). The analysis found a great diversity of tests and systems used for in vitro assays; many did not meet criteria for a valid test, and/or did not use validated cells and methods in the Organization for Economic Co-operation and Development Test Guidelines, and so these results could not be interpreted. In vivo assays were less common but better performed. It was not possible to develop conclusions on test system agreement, NM activity, or mechanism of action. However, the limited responses observed for most NMs were consistent with indirect genotoxic effects, rather than direct interaction of NMs with DNA. We propose a revised genotoxicity test battery for NMs that includes in vitro mammalian cell mutagenicity and clastogenicity assessments; in vivo assessments would be added only if warranted by information on specific organ exposure or sequestration of NMs. The bacterial assays are generally uninformative for NMs due to limited particle uptake and possible lack of mechanistic relevance, and are thus omitted in our recommended test battery for NM assessment. Recommendations include NM characterization in the test medium, verification of uptake into target cells, and limited assay-specific methods alterations to avoid interference with uptake or endpoint analysis. These recommendations are summarized in a Roadmap guideline for testing.