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

Abstract

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.

Genotoxic effects of silver nanoparticles with/without coating in human liver HepG2 cells and in mice

“…In the in vivo micronucleus experiment, the genotoxic effect of nanosilver was less than that in the in vitro experiment, These differences could be the fact that the body has an immune system and self‐repairing function, while cells cultured in vitro do not. In addition, evidence should be provided that the nanomaterials reached the target tissue in any in vivo assays (Elespuru et al, ). Our study lacked an analysis of uptake into the target tissue (bone marrow).…”

Genotoxicity of Nanomaterials: Advanced In Vitro Models and High Throughput Methods for Human Hazard Assessment—A Review

“…γH2AX is one of the DNA damage response proteins that accumulate and/or are modified in the vicinity of a chromosomal DNA DSB to form microscopically visible, subnuclear foci, contributing to their repair [ 127 ]. A number of studies have proposed C-termini phosphorylated histone protein, γH2AX, as a potential biomarker of DNA DSBs caused by genotoxicants [ 67 , 129 , 130 , 131 ]. This method is widely used in different fields including in vitro toxicology testing of environmental pollutants [ 132 ].…”

EURL ECVAM Genotoxicity and Carcinogenicity Database of Substances Eliciting Negative Results in the Ames Test: Construction of the Database

“…In the case of the latter, in several studies giving discordant results, the tested extracts were isolated either from dried milled roots or from leaves, and contained uncharacterised concentrations of the active pyrrolizidine alkaloid symphitine. Titanium oxide and C60 nanoparticles were excluded based on the recent work from the Health and Environmental Sciences Institute’s Genetic Toxicology Technical Committee (HESI-GTTC), which indicated that the Ames test is not recommended for genotoxicity assessment of nanomaterials [ 12 ].…”