Graphene-based materials (GBMs) are a broad family of novel carbon-based nanomaterials with many nanotechnology applications. The increasing market of GBMs raises concerns on their possible impact on human health. Here, we review the existing literature on the genotoxic potential of GBMs over the last ten years. A total of 50 articles including in vitro, in vivo, in silico, and human biomonitoring studies were selected. Graphene oxides were the most analyzed materials, followed by reduced graphene oxides. Most of the evaluations were performed in vitro using the comet assay (detecting DNA damage). The micronucleus assay (detecting chromosome damage) was the most used validated assay, whereas only two publications reported results on mammalian gene mutations. The same material was rarely assessed with more than one assay. Despite inhalation being the main exposure route in occupational settings, only one in vivo study used intratracheal instillation, and another one reported human biomonitoring data. Based on the studies, some GBMs have the potential to induce genetic damage, although the type of damage depends on the material. The broad variability of GBMs, cellular systems and methods used in the studies precludes the identification of physico-chemical properties that could drive the genotoxicity response to GBMs.

Graphene-based materials may pose a potential risk for human health due to occupational exposure, mainly by inhalation. This study was carried out on bronchial epithelial 16HBE14o− cells to evaluate the role of chemical reduction and formulation of graphene oxide (GO) on its cytotoxic potential. To this end, the effects of GO were compared to its chemically reduced form (rGO) and its stable water dispersion (wdGO), by means of cell viability reduction, reactive oxygen species (ROS) generation, pro-inflammatory mediators release and genotoxicity. These materials induced a concentration-dependent cell viability reduction with the following potency rank: rGO > GO >> wdGO. After 24 h exposure, rGO reduced cell viability with an EC₅₀ of 4.8 μg/mL (eight-fold lower than that of GO) and was the most potent material in inducing ROS generation, in contrast to wdGO. Cytokines release and genotoxicity (DNA damage and micronucleus induction) appeared low for all the materials, with wdGO showing the lowest effect, especially for the former. These results suggest a key role for GO reduction in increasing GO cytotoxic potential, probably due to material structure alterations resulting from the reduction process. In contrast, GO formulated in a stable dispersion seems to be the lowest cytotoxic material, presumably due to its lower cellular internalization and damaging capacity.

A new material composed of a kaolin base with silver nanoparticles (AgNPs) attached to its surface was developed, as an alternative to antibiotics used as supplements in animal feed. As part of its safety assessment, an in vivo geno-toxicological evaluation of this material was conducted in rats. First, a preliminary dose finding study was carried out to decide the doses to be tested in the main study: 50, 300 and 2000 mg/kg b.w. For the main study, a combined strategy composed of the MN test (TG 474) and the comet assay (TG 489), integrated in a repeated dose 28-day oral toxicity study (TG 407), was performed. A No Observed Adverse Effect Level (NOAEL) of 2000 mg of the silver-kaolin formulation/kg b.w. by oral route, for 28 days, was determined. The silver-kaolin formulation did not induce micronuclei in bone marrow, or DNA strand breaks (SBs) or alkali labile sites (ALS) in liver, spleen, kidney or duodenum at any dose. The modified Fpg comet assay did not reveal oxidized bases in the same tissues at the dose of 2000 mg/kg b.w. Silver was quantified by ICP-MS in all the target organs, confirming the negative results obtained under these conditions.