In-vitro analysis finds boron nitride and graphene oxide could irreparably damage DNA

The market for 2D materials is booming. Molybdenum disulfide (MoS2), boron nitride (hBN), and a whole host of graphene-related materials are finding widespread use in commercial products. Their unique properties and ever-decreasing manufacturing costs is seeing them play increasingly important roles in sectors like biomedicine and energy storage. However, there are still some unanswered questions with regards to the toxicological risk that these 2D materials might pose to human health. While this has prompted international bodies like the OECD and the EU to develop standardised test methods for cyto- and genotoxic effects, such existing standards aren’t optimised for all 2D materials (2D-NMs).

Writing in Carbon [DOI: 10.1016/j.carbon.2023.118426], Spanish researchers report on a reliable modification of a specific in vitro test method that they say “…could help establish a new standard to compare all NMs and assess the genotoxicity of 2D-NM at low and high doses.” The test they’ve focused on is OECD TG 487; the “in vitro mammalian cell micronucleus test”, which detects genotoxic damage in cells through the presence of whole or fragmented chromosomes (known as micronuclei, MNu) formed during cell division.

The MNu assay has been widely studied for different nanomaterials, but the characteristics of graphene-related materials often impact the accuracy of the associated techniques used to analyse MNu. The central challenge to determining the risk to human health of 2D materials surrounds their tendency to form agglomerates, as this can lead to false-positive or false-negative outcomes. To tackle this, these researchers looked to dextran – a non-toxic additive that can increase the viscosity of a cell medium, thus improving the stability of materials suspended in it. They chose CHO-K1 cells – widely used in toxicology research – as their platform, and cultured them in dextran.

They then prepared and characterised samples of four 2D materials: graphene oxide (GO), few-layer graphene (FLG), MoS2, and hBN, before incubating each one at varying concentrations (0.05, 0.5, 5, 20, and 50 μg/ml), with the cells. They also introduced controlled shaking of the platform during the incubation phase. This was found to minimize agglomeration of the 2D materials (excluding hBN, which “did not dramatically agglomerate”), and prevented their rapid deposition onto the cells.

A concentration of 20 μg/ml of FLG and MoS2 was shown to reduce viability significantly (12.32% and 13.66%, respectively) compared to the control, and this effect was even more pronounced at higher concentrations. At 50 μg/ml, hBn also reduced viability significantly (~19%). In contrast, no significant changes were observed for GO at any dose. However, they write, “…we found that all the selected concentrations of NMs remained within the established threshold for cytotoxicity as defined by the OECD guidelines,” indicating a lack of significant cytotoxic effects.

Assessment of genotoxicity found a significant increase in the number of micronuclei in GO-treated cells at low concentrations (0.05–5 μg/ml). A similarly significant increase in micronucleate cells was also observed in cells exposed for 24 h to 0.05 μg/ml of hBN. No differences were observed for the other nanomaterials tested. This means that low doses of GO and hBN induced non-reversible chromosomal damage in CHO-K1 cells; making them genotoxic.

The authors conclude, “…. we demonstrated that it is feasible to increase the stability and dispersibility of NMs in a standard commercial cell culture media with controlled shaking…”. They say that their modified approach “…shows that GO and hBN are genotoxic at low concentrations, which causes irreparable DNA damage. These findings must be taken into consideration for the further use of these NMs.”


S. García-Carpintero, V. Jehová González, J. Frontiñán-Rubio, A. Esteban-Arranz, E. Vázquez, M. Durán-Prado. “Screening the micronucleus assay for reliable estimation of the genotoxicity of graphene and other 2D materials,” Carbon 215 (2023) 118426. DOI: 10.1016/j.carbon.2023.118426