Salma Achawi

Ludovic Huot

Fabrice Nesslany

Jérémie Pourchez

Sophie Simar

Valérie Forest
email: vforest@emse.fr

Bruno Feneon
email: bruno.feneon@michelin.com

Exploring Graphene-Based Materials' Genotoxicity: Inputs of a Screening Method

Keywords: Graphene-based materials, genotoxicity, high-throughput screening, structure-activity relationships

Graphene-based materials (GBMs) are promising nanomaterials, and several innovations depend on their use. However, the assessment of their potential hazard must be carefully explored before entering any market. GBMs are indeed well-known to induce various biological impacts, including oxidative stress, which can potentially lead to DNA damage. Genotoxicity is a major endpoint for hazard assessment and has been explored for GBMs, but the available literature shows conflicting results. In this study, we assessed the genotoxicity of 13 various GBMs, one carbon black and one amorphous silica through a DNA damage response assay (using a human respiratory cell model, BEAS-2B).

Concurrently, oxidative stress was assessed through a ROS production quantification (DCFH-DA assay using a murine macrophage model, RAW 264.7). We also performed a full physicochemical characterization of our samples to explore potential structureactivity relationships involving genotoxicity. We observed that surface oxidation appears linked to genotoxicity response and were able to distinguish several groups within our studied GBMs showing different genotoxicity results. Our findings highlight the necessity to individually consider each nanoform of GBMs since the tested samples showed various results and modes of action. We propose this study as a genotoxicity assessment using a high-throughput screening method and suggest few hypotheses concerning the genotoxicity mode of action of GBMs.

Introduction

Graphene-based materials (GBMs) are continuously raising interest since the isolation of graphene in 2004 [START_REF] Novoselov | Electric field effect in atomically thin carbon films[END_REF]. They include subfamilies such as graphene oxide (GO), reduced graphene oxide (RGO) or graphene nanoplatelets (GNP). Despite their promising properties, the industrial use of GBMs is still minor, and only few of them already led to commercialized innovations. This is partly due to uncertainties concerning their potential risk on human health.

GBMs' toxicity has often been questioned and thoroughly investigated [START_REF] Fadeel | Safety Assessment of Graphene-Based Materials: Focus on Human Health and the Environment[END_REF].

GBMs may indeed trigger various effects: inflammation [START_REF] Di Cristo | Repeated exposure to aerosolized graphene oxide mediates autophagy inhibition and inflammation in a three-dimensional human airway model[END_REF] or mitochondria physiology imbalance [START_REF] Jarosz | Oxidative Stress and Mitochondrial Activation as the Main Mechanisms Underlying Graphene Toxicity against Human Cancer Cells[END_REF] are only few examples. However, GBMs are mostly well-known to induce oxidative stress [START_REF] Chen | Oxidative stress and immunotoxicity induced by graphene oxide in zebrafish[END_REF][START_REF] Dziewięcka | Short-term in vivo exposure to graphene oxide can cause damage to the gut and testis[END_REF][START_REF] Vranic | Confocal and optical microscopy for internalization study. A: Optical microscopy of BEAS-2B and a micronucleus. B: Optical microscopy of BEAS-2B and a presumed RGO4 agglomerate. C: Focal plane number 8 (see figure 2) for BEAS-2B and micronucleus. D[END_REF][START_REF] Ain | The systemic effect of PEG-nGO-induced oxidative stress in vivo in a rodent model[END_REF]. Genotoxicity is a crucial toxicity endpoint, and two mechanisms can be highlighted: primary genotoxicity is due to the nanomaterial's interaction with target cells whereas secondary genotoxicity involves mechanisms in which nanomaterials do not interact directly with the target cell.

Primary genotoxicity can be direct or indirect. Direct genotoxicity is a consequence of a direct interaction between nanomaterial and DNA or chromosomes by direct binding with DNA or through mechanical effects. It is often considered that direct genotoxicity is a non-threshold effect, supposingly increasing the risk from the smallest dose [START_REF] Jenkins | Genotoxic thresholds, DNA repair, and susceptibility in human populations[END_REF]. GBMs such as graphene quantum dots or GO have been shown to be able to penetrate cell nucleus [START_REF] Ren | DNA cleavage system of nanosized graphene oxide sheets and copper ions[END_REF][START_REF] Liu | Graphene oxide can induce in vitro and in vivo mutagenesis[END_REF], a direct contact with nuclear DNA can then be suspected. Some GBMs are known to interact with specific loci of the chromatin [START_REF] Sun | Graphene oxide regulates cox2 in human embryonic kidney 293T cells via epigenetic mechanisms: dynamic chromosomal interactions[END_REF]). GO, for example, was found to bind directly DNA, which affects the replication process [START_REF] Liu | Graphene oxide can induce in vitro and in vivo mutagenesis[END_REF]. A direct interaction with DNA can also occur during interphase and may affect replication or transcription mechanisms, which can impact DNA structure and even cause cleavage [START_REF] Ren | DNA cleavage system of nanosized graphene oxide sheets and copper ions[END_REF]. Furthermore, a direct interaction can also be observed between the GBMs and chromosomes during mitosis: in that case, loss of chromosome (aneugenicity) or chromosomal break (clastogenicity) can be observed. Lastly, carbon-based materials are well-known to instrinsically generate free radicals: GBMs are of no exception. Depending on their surface parameters (such as surface oxidation and suface defects), free radicals are released from the GO surface and can directly damage DNA [START_REF] Dizdaroglu | Free radical-induced damage to DNA: mechanisms and measurement[END_REF].

Indirect genotoxicity does not directly affect DNA but can affect genetic information. GBMs can interact with nuclear proteins involved in the replication, repair or transcription processes [START_REF] Chatterjee | Differential genotoxic and epigenotoxic effects of graphene family nanomaterials (GFNs) in human bronchial epithelial cells[END_REF]. Oxidative stress is defined by the imbalance between pro-oxidants species (including reactive oxygen species, ROS and reactive nitrogen species, RNS) and antioxidants. The increase of ROS is considered to be a major step of the GBMs mode of action and can also indirectly lead to genotoxicity through various mechanisms such as DNA breakage or chromatin remodeling [START_REF] Tabish | Developing the next generation of graphenebased platforms for cancer therapeutics: The potential role of reactive oxygen species[END_REF])(see Figure 1). 8-oxo-dG, an oxidized derivative of deoxyguanosine (one of the deoxyribonucleosides composing DNA), can be formed by the attack of ROS on DNA and has been observed after a GO exposure (Gurunathan et al. 2019a(Gurunathan et al. , 2019b)). When treated with antioxidant (e.g., Nacetylcysteine), cells exposed to GBMs show greatly reduced genotoxicity (Burgum, Clift, Evans, Hondow, Tarat, et al. 2021). GBMs often present carboxyl groups on their surface which can generate free radicals, eventually inducing genotoxicity [START_REF] Burgum | In Vitro Primary-Indirect Genotoxicity in Bronchial Epithelial Cells Promoted by Industrially Relevant Few-Layer Graphene[END_REF]. Other redox-active groups can greatly affect GBMs's oxidative stress and ROS production [START_REF] Pieper | Endoperoxides Revealed as Origin of the Toxicity of Graphene Oxide[END_REF]. GBMs can also induce mitochondria stress which can also lead to increase the cellular ROS production [START_REF] Jaworski | Degradation of Mitochondria and Oxidative Stress as the Main Mechanism of Toxicity of Pristine Graphene on U87 Glioblastoma Cells and Tumors and HS-5 Cells[END_REF]. Moreover, some GBMs can inhibit the antioxidant enzymes (including catalase and superoxide dismutase), which obviously cause an increase in oxidative stress [START_REF] Deng | Nitrogen-doped graphene quantum dots (N-GQDs) perturb redoxsensitive system via the selective inhibition of antioxidant enzyme activities in zebrafish[END_REF]. In physiological conditions and until a certain point, natural cellular mechanisms will compensate this aggression, explaining why indirect genotoxicity is a thresholded effect. Under a determined threshold, the defense mechanisms are still operational and no adverse effect is actually induced [START_REF] Kirsch-Volders | Indirect mechanisms of genotoxicity[END_REF].

Secondary genotoxicity involves mechanisms in which nanomaterials do not interact directly with the target cell but produce an inflammatory response in neighboring cells which will generate ROS/RNS and induce a (secondary) genotoxic effect in the target cell [START_REF] Evans | Critical review of the current and future challenges associated with advanced in vitro systems towards the study of nanoparticle (secondary) genotoxicity[END_REF]. Hence, secondary genotoxicity is mainly driven by an inflammatory response (Åkerlund et al. 2019).

Briefly, aneugenicity, clastogenicity or mutagenicity are major genotoxicity mechanism. For aneugenicity, genotoxicants act primarily on non-DNA targets (microtubule, centrosome or kinetochore [START_REF] More | Guidance on aneugenicity assessment[END_REF])) or cause damage to the mitosis apparatus, leading to improper chromosome segregation [START_REF] Parry | The detection and evaluation of aneugenic chemicals[END_REF][START_REF] Parry | Detection and characterization of mechanisms of action of aneugenic chemicals[END_REF]. For clastogenicity, structural chromosome aberrations such as chromatid/ chromosome breaks occur [START_REF] Bignold | Mechanisms of clastogen-induced chromosomal aberrations: a critical review and description of a model based on failures of tethering of DNA strand ends to strand-breaking enzymes[END_REF]. Clastogenic agents can covalently bind to DNA or enzymes, leading to chromosome breakage. Mutagenicity corresponds to the induction of DNA mutations [START_REF] Kumar | Mutagenicity: Assays and Applications[END_REF], either by direct interaction with DNA or chromatin or by indirect mechanisms, such as through generation of reactive oxygen species or inflammation (DeMarini 2019). Genotoxicity is associated to serious health effects, the first one being cancer [START_REF] Phillips | Genotoxicity: damage to DNA and its consequences[END_REF]: some genotoxic agents can indeed cause mutations that can eventually lead to malign tumor. Hence, most carcinogenic chemicals are genotoxic [START_REF] Hayashi | Overview of genotoxic carcinogens and non-genotoxic carcinogens[END_REF], which make the measurement of this endpoint critical for hazard assessment.

The most common in vivo genotoxicity assays are chromosome aberration, micronucleus and comet assays (OECD 473,474 and 489,respectively) [START_REF] Magdolenova | Mechanisms of genotoxicity. A review of in vitro and in vivo studies with engineered nanoparticles[END_REF]. However, in a context of reduction, refinement and replacement of animal testing, and given the considerable number of nanomaterials to be tested, the scientific community is questioning a systematic use of in vivo models, especially considering that in vitro genotoxicity assessment can also be reliable when optimized [START_REF] Corvi | In vitro genotoxicity testing-Can the performance be enhanced? Food and Chemical Toxicology[END_REF]. In the context of a REACH registration of a substance, genotoxicity is currently measured with a mutation test on bacterial system (Ames test, OECD 471) [START_REF] Stead | Modeling the Ames test[END_REF]) and an in vitro genotoxicity assay on mammalian cell cultures (e.g., gene mutation assay and/or a chromosomal aberrations test). However, it has been demonstrated that the Ames' test is not optimal for nanoforms [START_REF] Doak | In vitro genotoxicity testing strategy for nanomaterials and the adaptation of current OECD guidelines[END_REF]) and can potentially be replaced by a mutation test on in vitro mammal models (OECD guideline 476 or 490). Otherwise, the micronucleus test described in OECD guideline 487 (OECD 2016) is well adapted for nanomaterials but an adaptation is necessary [START_REF] Gonzalez | Adaptations of the in vitro MN assay for the genotoxicity assessment of nanomaterials[END_REF]. The increasing number of nanomaterials [START_REF] Inshakova | World market for nanomaterials: structure and trends[END_REF], is urging us to rethink the methodology of genotoxicity assessment. More and more industries integrate sustainability and safety to their research and development, which implies to consider the use of a safe-by-design approach [START_REF] Schwarz-Plaschg | Making Nanomaterials Safer by Design?[END_REF]. In this respect, high throughput screening (HTS) genotoxicity testing can present lots of advantages [START_REF] Sukumaran | High-throughput approaches for genotoxicity testing in drug development: recent advances[END_REF].

Overall, a HTS method provides a quick overview of a toxic mode of action. This implies the coordinated measurement of multiple biomarkers on the same samples. Few DNA damage response (DDR) biomarkers exist and some of them have been thoroughly studied and validated. This is the case of the γH2AX histone [START_REF] Kopp | Validation of the γH2AX biomarker for genotoxicity assessment: a review[END_REF] as well as the H3 histone phosphorylation [START_REF] Khoury | Complementarity of phosphorylated histones H2AX and H3 quantification in different cell lines for genotoxicity screening[END_REF][START_REF] Hamilton | The characterization of γH2AX and p53 as biomarkers of genotoxic stress in a rainbow trout (Oncorhynchus mykiss) brain cell line[END_REF]. γH2AX is sensitive to double strand DNA breakage [START_REF] Rogakou | DNA doublestranded breaks induce histone H2AX phosphorylation on serine 139[END_REF][START_REF] Bonner | GammaH2AX and cancer[END_REF][START_REF] Kinner | Gamma-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin[END_REF]) which makes it a biomarker of clastogenicity [START_REF] Matsuzaki | Whole cell-ELISA to measure the gammaH2AX response of six aneugens and eight DNA-damaging chemicals[END_REF], especially when combined with phospho-histone H3 detection [START_REF] Mishima | Chromosomal aberrations, clastogens vs aneugens[END_REF] which is itself a biomarker of aneugenicity (Wilde et al. 2020). γH2AX is also a biomarker of mutagenicity if we consider the repair mechanisms that can be involved after interaction with DNA. The endpoint p53 as an indicator of nuclear p53 may also be investigated. Briefly, p53 is a tumor suppressor and, when a mutation is detected in a cell, it can stop the cell cycle to trigger DNA repair or induce apoptosis of the cell carrying the mutation. It is a biomarker used for detecting genotoxicity [START_REF] Bhaskaran | Fish p53 as a possible biomarker for genotoxins in the aquatic environment[END_REF] or even cancer [START_REF] Hayman | What is the potential of p53 isoforms as a predictive biomarker in the treatment of cancer?[END_REF]. In this paper, we used a HTS method for measuring the genotoxicity impact of a various GBMs. We also studied the relationship between surface oxidation and genotoxicity outcome and discussed potential GBMs genotoxicity mechanisms.

Material and Methods

Nanomaterials

We tested 13 various GBMs. For comparison, we also tested a carbon black and an amorphous silica as reference materials. These samples are depicted in Table 1. Their specific surface area (SSA) was determined by the BET technique (adsorption of nitrogen, with degassing system Micromeritics). Their surface oxidation was determined with XPS (X-ray Photo spectroscopy, Quantera Scanning XPS microprobe, Physical Electronics). Lateral size was determined with electron microscopy (Field Emission Scanning Electron Microscope, from JEOL). The full physicochemical characterization of each of these samples (including ICP analysis, full XPS characterization and RAMAN spectrum) can be found in supplementary data 1.

All nanomaterials were prepared and dispersed in deionized water at 1600 µg/mL as stock solution. Successive dilutions were then prepared in 10% fetal calf serum Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) supplemented with 1% penicillin-streptomycin (Sigma-Aldrich) to obtain the following range of final doses: 100, 75, 50, 37.5, 25, 8.33 and 4.17 µg/cm².

DDR assay

Cell culture and culture medium

The human bronchial epithelial cell line BEAS-2B was obtained from the American Type Culture Collection (ATCC® CRL-9609™). BEAS-2B cells were seeded at a density of 5x10 5 cells in 75 cm² Corning® CellBIND® surface cell culture flasks (Corning) and maintained at 37°C in a humidified 5% CO2 atmosphere in complete Pneumacult Ex-medium (Stemcell), a Medium was renewed every 2 to 3 days and cells were subcultured before reaching confluence since confluent cultures rapidly undergo squamous terminal differentiation. The doubling time of BEAS-2B cells is approximately 24 hours.

Cell exposure to nanomaterials

A cell suspension at a density of 2x10 5 cells/mL was prepared in complete Pneumacult Exmedium. A volume of 100 µL of cell suspension was distributed in 96-well plates CellBIND® surface (Costar) then incubated at 37°C with 5 ± 0.5 % CO2, 95 ± 5 % humidity. Two treatment schedules were carried out: a 4-hour short-term treatment and a 24-hour continuous treatment.

The treatment consisted in replacement of the medium by 200 µL of each dilution of the different nanomaterials prepared in the Complete Pneumacult Ex-treatment medium. Then, the plates were incubated at 37 °C with 5 ± 0.5 % CO2, 95 ± 5 % humidity. Duplicate cell cultures treated with the solvent were used as negative controls. Methylmethane sulfonate (300 µM; Merck, clastogenic substance), vinblastine (24 nM; Merck, aneugenic substance) and griseofulvin (30 µg/mL, Merck, aneugenic substance) were used as positive controls. Please note that for each experiment, a significant genotoxic effect was observed. At the end of the treatment (4 or 24 hours), the cells were washed by addition of 200 µL PBS1X (Gibco) at 37°C. Then, the washing buffer was replaced by a volume of 50 µL of pre-warmed TrypLE Express (Gibco) at 37°C. Plates were then incubated for 5 minutes at 37°C.

DDR assay protocol

The MultiFlow DNA Damage Kit -p53, γH2AX, Phospho-Histone H3 Kit from Litron Laboratories (product code MultiFlow (p53,γH2AX, H3) ; Rochester, NY 14623, USA) was used. The DDR is a HTS assay that allows the simultaneous measurements of genotoxicity biomarkers (H2AX and PH3) as well as p53, which can be considered as a biomarker for nuclear translocation. Moreover, the flow cytometry allows to assess cytotoxicity through the nuclei count. Please note that when the nuclei count was considered too low, an additional analysis was performed. Overall, the tested concentration did not induce a major cytotoxicity (the relative nuclei count was largely higher than 50% for moderate doses) on BEAS-2B cells.

The experiments were conducted upon the protocol provided by Litron Laboratories, as follows:

Labelling: A volume of 50 µL of the "Complete Labelling Solution" (containing Nuclei Release Solution, Counting Beads, DNA Stain, RNase Solution, filtered FBS and antibodies) was added to individual wells of a clean 96 U-bottom well plate. The cells from the treatment plates were gently resuspended and a volume of 25 µL was transferred in the plate containing Complete Labelling Solution. The mix was incubated for 30 minutes at room temperature, protected from light. After incubation, analysis was carried out in flow cytometry.

Flow Cytometric Data Acquisition: Analysis was performed on at least 20 µL per well mixed (setting of the cytometer with 4 cycles of mixing with volumes of 40 µL), at a rate of 1 µL/sec. Analysis: Briefly, analysis was done following the global Evaluation Factor (GEF) which indicates a significant increase in mutant phenotype cell and is based on the distribution of the negative control mutation frequency data from several proficient laboratories [START_REF] Moore | Mouse lymphoma thymidine kinase gene mutation assay: follow-up meeting of the International Workshop on Genotoxicity Testing--Aberdeen, Scotland, 2003--Assay acceptance criteria, positive controls, and data evaluation[END_REF][START_REF] Bryce | Interlaboratory evaluation of a multiplexed high information content in vitro genotoxicity assay[END_REF].

 A test item is concluded clastogen if 2 successive doses meet or exceed the GEF for at least two out of the 2 clastogen-sensitive biomarkers,  A test item is concluded aneugen if 2 successive doses meet or exceed the GEF for at least two out of the 2 aneugen-sensitive biomarkers,  In case where both clastogen and aneugen-sensitive biomarkers exceeded GEF(s), the category with the greater number of significant biomarkers is selected as the predominant mode of action,  When less than 2 clastogen-and aneugen-sensitive biomarkers meet or exceed the GEFs, the call is non-genotoxic under the test conditions used.

DCFH-DA assay

The cell model used in the assay was RAW264.7 murine macrophage cell line, provided by ATCC Cell Biology Collection (Promochem LGC). It derived from mice peritoneal macrophages transformed by the Albeson Murine Leukemia Virus. Cells were grown in 10% fetal calf serum Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) supplemented with 1% penicillin-streptomycin (Sigma-Aldrich) and maintained at 37°C under a 5% carbon dioxide humidified atmosphere. It was then seeded in 96-well plates the day before exposure. Cells were then exposed to GBMs with exposure doses from 8 to 75 µg/cm² for 24 hours. ROS production was measured through OxiSelect™ Intracellular ROS Assay Kit (STA-342) as previously reported (Achawi et al. 2021a).

Results

GBMs internalization in BEAS-2B

We performed an exploratory internalization study, presented in supplementary data 2. We exposed BEAS-2B cells to RGO4 (GBM sample showing a large mean lateral size of 31.6 µm) and used optic and confocal microscopy for observation, and it seems that RGO4 can potentially be internalized in BEAS-2B cells. Although these findings are not necessarily applicable to the rest of the samples, they show that one of our largest GBM appears to be internalized in BEAS-2B cells.

DDR results

The expression of each biomarker indicating a potential aneugenic or clastogenic effect is presented in Table 2.

Six samples did not show any genotoxic effect: CB, GNP9, RGO4, RGO5, RGO7 and RGO9.

Five are aneugenic only: GNP2, GNP3, GNP7, GNP8 and amorphous silica. 2 samples are aneugenic and show equivocal clastogenicity/mutagenicity (RGO3 and RGO8): indeed, they do not present two successive doses leading to a positive response for both exposure times (4h and 24h) (see section 2.2.3) but still present signs of positivity. 2 samples are both aneugenic and clastogenic: GNP1 and RGO6.

It appears that the GBMs are most likely to show aneugenic than clastogenic effects. The exposure to GBMs does not increase the p53 biomarker, except for one sample (RG08). It is worth noting that the sample that activates p53 is also aneugenic and potentially mutagenic/clastogenic.

Overall, a majority of our GNPs showed aneugenicity while a minority of them showed clastogenicity / mutagenicity.

When we obtained a positive result for genotoxicity, we usually observed a dose-dependent response.

Potential correlation between surface oxidation and genotoxicity

We tested potential correlation between physicochemical characteristics and genotoxicity impact and underscored the effect of surface oxidation. In Figure 4, we present the surface oxidation of samples grouped by their genotoxicity response (no response in green, aneugenic only in orange and both response in red), and the mean surface oxidation of their group (hatched).

GBM samples having a mean surface oxidation of 5% demonstrated no genotoxicity signal.

The samples showing only an aneugenic mode of action have a 6% mean surface oxidation whereas the samples that show high genotoxicity have a 12% of mean surface oxidation, as shown in Figure 2.

It appears that the clastogenicity /mutagenicity phenomenon is rather linked to a high level of surface oxidation: this is particularly obvious for RGOs, but this observation might not be applicable to GNPs (GNP1 shows aneugenicity and mutagenicity/clastogenicity while having a low surface oxidation).

We also investigated the potential relationship between the metal contents and genotoxicity and did not observe any correlation.

ROS production and genotoxicity

We tested the relationship between the results of the genotoxicity screening test presented above and the ROS production induced by each sample (as previously assessed by the DCFH-DA assay on RAW264.7 cell model, exposed for 24 hours to the nanomaterials). The results are presented in Figure 3.

Noteworthy, despite the cell models were different (BEAS-2B for DDR assay and RAW264.7 for DCFH-DA assay), the exposure concentrations used for the two assays were consistent (i.e. from 8.1 µg/cm² to 75 µg/cm² for DCFH-DA assay and from 4.1 µg/cm² to 100 µg/cm² for DDR screening assay). Please note that samples RGO3 and RGO8 were considered to be equivocal for clastogenicity / mutagenicity (see Table 2). For an easier reading of the results, we classified these two samples as positive for clastogenicity / mutagenicity in this Figure .   For ROS production, a sample was considered positive when at least two exposure concentrations led to a significant response when compared to negative control (unexposed cells). Note that all positive GBMs for ROS production led to a significant response from the lower dose (8.1 µg/cm²), except for RGO3 which led to a significant response at a moderate dose (16 µg/cm²).

It appears that some GBMs samples have the same outcome for ROS production and aneugenicity: one sample (GNP9) does not show any effect for both assays and 6 samples (GNP1, RGO3, RGO8, RGO6, GNP2, GNP3) are positive for both assays. However, 5 GBMs lead to an increased ROS production without leading to the detection of aneugenicity or clastogenicity (RGO4, RGO5, RGO7, RGO9 and CB) and 3 samples (GNP7, GNP8 and amorphous silica) are aneugenic without causing ROS production.

Clastogenicity is negative for most of our samples. However, when samples are clastogenic (4 samples: GNP1, RGO3, RGO8 and RGO6), they are also aneugenic and increase cellular ROS production. These samples have respectively a SSA of 545, 283, 270 and 440 m²/g, a lateral size of 8.4, 1.3, 32 and 1.0 µm and a surface oxidation of 11.9, 3.2, 15.9 and 17.2 %.

The only sample that does not cause ROS increase, clastogenicity and aneugenicity is GNP9. This sample, as shown in Table 2, has a low SSA (119 m²/g), a very large mean lateral size (38.6 µm) and a moderate surface oxidation (5.7 %).

Discussion

GBMs' genotoxicity: our results and the literature

In vitro studies

In this study, we tested the clastogenicity/mutagenicity and aneugenicity of 13 GBMs (including GNPs and RGOs), one carbon black and one amorphous silica on BEAS-2B cells.

Carbon black is negative for both genotoxicity assays and the amorphous silica is aneugenic only. Overall, GBMs are more often aneugenic than clastogenic or mutagenic: among 13 GBMs, 8 of them are aneugenic whereas only 4 (2 of them being equivocal) are clastogenic.

None of the tested samples is only clastogenic: the clastogenic samples (RGO3, RGO6, RGO8 and GNP1) are also aneugenic and cause increased ROS production. In this regard, we can probably rule out a direct clastogenicity mechanism. Some GBMs appear to have a high genotoxic impact even at quite low doses, this is not completely surprising as some of these samples also show a very high impact for other in vitro studies performed on RAW 264.7 (Achawi et al. 2021a) and on an acellular FRAS assay (Achawi et al. 2021b).

Carbon blacks are globally considered to be non-genotoxic [START_REF] Chaudhuri | Evaluating the evidence on genotoxicity and reproductive toxicity of carbon black: a critical review[END_REF]. This is in agreement with our results showing no noticeable genotoxicity of our moderate specific surface area carbon black. The aneugenicity of amorphous silica has been highlighted in the work of Gonzalez et al. [START_REF] Gonzalez | Induction of chromosome malsegregation by nanomaterials[END_REF][START_REF] Gonzalez | Amorphous silica nanoparticles alter microtubule dynamics and cell migration[END_REF] and is confirmed in our study.

Concerning GBMs, literature data tend to show mixed results: GO have mostly been considered as genotoxic [START_REF] Mohamed | Estimation of genomic instability and mutation induction by graphene oxide nanoparticles in mice liver and brain tissues[END_REF][START_REF] Mohamed | Induction of chromosomal and DNA damage and histological alterations by graphene oxide nanoparticles in Swiss mice[END_REF]. Their impact can be lowered with functionalization [START_REF] Liu | Graphene oxide can induce in vitro and in vivo mutagenesis[END_REF]) and smallest GO tend to have more genotoxic effects [START_REF] De Marzi | Flake size-dependent cyto and genotoxic evaluation of graphene oxide on in vitro A549, CaCo2 and vero cell lines[END_REF].

Another study shows that reduction enhances DNA damage caused by RGO on human retinal pigment epithelium cells [START_REF] Ou | Oxygen content-related DNA damage of graphene oxide on human retinal pigment epithelium cells[END_REF]). The size is also a critical parameter: the smallest RGOs are the most genotoxic whereas large ones only show a low effect [START_REF] Akhavan | Size-dependent genotoxicity of graphene nanoplatelets in human stem cells[END_REF]).

On BEAS-2B, GNPs also show a genotoxic impact (comet assay) even when functionalized (COOH and NH2) [START_REF] Chatterjee | Differential genotoxic and epigenotoxic effects of graphene family nanomaterials (GFNs) in human bronchial epithelial cells[END_REF]. Lastly, few layers graphenes (FLG) were tested on bronchial epithelial cells: the neutral and the amine-FLG cause genotoxicity whereas the carboxyl-FLG do not induce genotoxicity [START_REF] Burgum | In Vitro Primary-Indirect Genotoxicity in Bronchial Epithelial Cells Promoted by Industrially Relevant Few-Layer Graphene[END_REF]).

Gurcan et al. [START_REF] Gurcan | A closer look at the genotoxicity of graphene based materials[END_REF]) reviewed the GBM's genotoxicity modes of action: the indirect genotoxicity through the increase of ROS is particularly highlighted. To address the potential genotoxicity of GBMs, we selected few papers presenting in vitro investigation of GBMs' genotoxicity (presented in Table 3) [START_REF] Akhavan | Size-dependent genotoxicity of graphene nanoplatelets in human stem cells[END_REF], 2013[START_REF] Li | Surface coating-dependent cytotoxicity and degradation of graphene derivatives: towards the design of non-toxic, degradable nano-graphene[END_REF][START_REF] Bengtson | No cytotoxicity or genotoxicity of graphene and graphene oxide in murine lung epithelial FE1 cells in vitro[END_REF][START_REF] Hashemi | Synthesis and cyto-genotoxicity evaluation of graphene on mice spermatogonial stem cells[END_REF][START_REF] Mittal | Physico-chemical properties based differential toxicity of graphene oxide/reduced graphene oxide in human lung cells mediated through oxidative stress[END_REF][START_REF] Arbo | In vitro cardiotoxicity evaluation of graphene oxide[END_REF][START_REF] Jia | Nanotoxicity of different sizes of graphene (G) and graphene oxide (GO) in vitro and in vivo[END_REF].

Note that methodological aspects need to be considered when comparing these results. Firstly, the tested GBMs have different physicochemical characteristics. Their exposure concentrations also vary; most papers exposed cells to several doses up to 100 µg/mL, whereas the article [START_REF] Li | Surface coating-dependent cytotoxicity and degradation of graphene derivatives: towards the design of non-toxic, degradable nano-graphene[END_REF]) tested only one high exposure concentration (200 µg/mL). Moreover, the cell models differ, which can impact the measured genotoxicity. A total of 26 different GBMS were studied within these 8 studies. Tested GBMs were GOs and RGOs, functionalized in some cases with BSA and PEG and the most common investigated endpoint was the primary DNA fragmentation through the comet assay. Only 5 GBMs did not show genotoxicity while 21 of them showed moderate to strong in vitro genotoxicity. 13 of these samples were RGOs and among them, 8 showed potential for DNA fragmentation and the remaining 5 showed none, or only at very high dose and long exposure.

These results slightly differ from ours: we observed that most of our RGO samples do not show genotoxicity signs: neither clastogenicity / mutagenicity nor aneugenicity occurs, and only 3 of them are positive for both aneugenicity and clastogenicity. Our GNPs appear quite reactive for aneugenicity: 5 of them are positive but only one is both aneugenic and clastogenic. The comparison of the published results and ours results is delicate: our DDR testing allows specifically investigating the aneugenicity and the mutagenicity / clastogenicity mechanisms whereas DNA fragmentation highlighted with comet assay does not necessarily imply a clastogenicity: pH variation can also affect the DNA fragmentation. Moreover, comet assay does not detect aneugenicity.

In vivo studies

In vivo studies tend to show different results concerning GBMs genotoxicity and its potential mode of action. Weekly exposure of male mice to GO by intraperitoneal injections for up to 8 weeks resulted in DNA fragmentation in lung as well as structural chromosomal aberrations in bone marrow [START_REF] El-Yamany | Graphene oxide nanosheets induced genotoxicity and pulmonary injury in mice[END_REF]). On the contrary, when Sprague-Dawley rats were exposed (nose-only respiratory exposure) to GNP in a 28-day study no DNA damage was found [START_REF] Kim | 28-Day inhalation toxicity of graphene nanoplatelets in Sprague-Dawley rats[END_REF]. However, in mice exposed to GO and RGO by single intratracheal instillation, an important inflammation was observed as well as DNA damage [START_REF] Bengtson | Differences in inflammation and acute phase response but similar genotoxicity in mice following pulmonary exposure to graphene oxide and reduced graphene oxide[END_REF]. Even if the authors did not make such assumptions, these results could indicate that inflammation is partially interfering with the observed DNA fragmentation. Studies using cell co-culture allowed to confirm that some GBMs could induce secondary genotoxicity through an inflammatory reaction [START_REF] Burgum | Vitro Lung Models to Assess the Mechanistic Genotoxicity of Characterised Few-Layer Graphene[END_REF]. These studies used different rodent models and have distinct route, protocol and dose of exposure. Moreover, the GBMs tested are GO and GNP.

Hence, comparing these results is delicate.

Genotoxicity and surface oxidation: a potential correlation

In Figure 2, we discussed the GBMs potential correlation between surface oxidation and genotoxicity (aneugenicity or mutagenicity/clastogenicity). It appears clearly that the negative samples for both aneugenicity and clastogenicity/mutagenicity endpoints have a low surface oxidation (mean 5 ± 1.9%) whereas samples that are clastogenic/mutagenic and aneugenic have a significantly higher surface oxidation (mean 12.1 ± 4.5 %). It hence appears that a higher surface oxidation is linked to clastogenicity/mutagenicity. This might be explained by the fact that, among other physicochemical characteristics such as size, shape or specific surface area, surface chemistry has an important impact on several genotoxicity mechanisms.

A high oxidation is not supposed to facilitate a direct interaction with DNA: the negative charge and usually higher surface defects of GOs can be a problem for a tight fixation [START_REF] Liu | DNA adsorbed on graphene and graphene oxide: Fundamental interactions, desorption and applications[END_REF].

As an example, GO and RGO interaction with DNA was compared, with a better adsorption for RGO [START_REF] Lu | Comparison of Graphene Oxide and Reduced Graphene Oxide for DNA Adsorption and Sensing[END_REF]. Hence, even if some samples are small enough to enter the cell and even the nucleus, we cannot assume that a higher surface oxidation allows a direct contact between DNA and GBMs: the observed clastogenicity might be due to another mechanism.

Oxidized surface reactivity might involve more reactive groups that could generate free radicals. It has indeed been observed previously that a modulation of surface oxidation could change the catalytic activity of graphene and graphite oxide: for producing free radicals, a sufficient oxidation is needed (Komeily-Nia et al. 2020).

Combining DDR testing and ROS production measurement can inform on genotoxicity mechanism

We investigated the relationship between ROS production and genotoxicity: these assays are both in vitro assays but were performed on different cell models: RAW264.7 cells were used for the DCFH-DA assay (ROS production) and BEAS-2B cells were used for DDR assay (clastogenicity or mutagenicity and aneugenicity). A large majority (11 out of 15) of the tested samples cause an increase of ROS production after an exposure to low to moderate doses (8 to 16 µg/cm²). A majority of the GBMs that increased ROS production also caused a positive response during genotoxicity testing, which may confirm that, for GBMs, genotoxicity is likely an indirect mechanism linked to oxidative stress. An interesting way to confirm this hypothesis would be to perform the same experiments with the adjunction of an antioxidant.

We observe that few samples increase the ROS production but do not induce genotoxicity. It might be possible that for these samples, the increased ROS production might be controlled by the cellular antioxidant defense and, under a certain threshold, will not cause indirect genotoxicity. Similarly, the CB showed an increased ROS production, with no genotoxicity, possibly indicating that at the tested doses, the antioxidant defenses were able to avoid indirect genotoxicity.

The amorphous silica shows a different pattern: no ROS production but still an aneugenic effect.

Silica's aneugenic effects has been thoroughly studied by Kirsch-Volders team, and was found to be mostly linked to chromosome migration alteration through interaction with microtubules and only minimally to the ROS production [START_REF] Gonzalez | Induction of chromosome malsegregation by nanomaterials[END_REF]. Interestingly, two GBM samples (GNP7 and GNP8) seem to show the same mechanism. For these two samples, the aneugenic impact might not be linked to oxidative stress. It is worth noting that these samples measure respectively 10.9 and 17.4 µm which probably makes these samples too large to have nuclear interaction with DNA. It might be interesting to investigate these specific GBMs' mode of action and compare it to amorphous silica's one. The other sample (GNP9) that has no impact on ROS production does not show genotoxicity either.

These results highlight that within the same family of nanomaterials, the genotoxicity mechanisms might vary. As noted previously, the genotoxicity (and its mode of action) is crucial for hazard assessment. Combining the results of genotoxicity and oxidative stress measurement can be an interesting approach to have a better insight on the genotoxicity mode of action. However, these conclusions must take into account a limitation of this study: the cell models used for each assay were different. RAW264.7 cells are murine macrophages whereas BEAS-2B cells are human epithelial respiratory cells: their cellular system might differ as well as their antioxidant capacity. Testing genotoxicity impact of GBMs in presence of antioxidant could help having a confirmation that the mechanism is oxidative stress related for some samples.

Discussing genotoxicity GBMs mechanisms

In Figure 4, we highlight the hypothetical mechanisms leading or not to in vitro genotoxicity among the tested GBMs.

We integrated the physicochemical characteristics possibly being involved, particularly surface oxidation (see section 4.2) and the information concerning ROS production caused by GBMs.

We can spot five groups of materials that appear to have different outcomes for genotoxicity and various modes of action:

1. No detectable genotoxicity or ROS production (GNP9): this sample induces neither increase in ROS production nor genotoxicity. The very large lateral size (38.6 µm) may prevent this substance from penetrating the cells combined with the low surface oxidation of this sample (5.7%) which might not cause the production of free radicals.

2. Increased ROS production as well as aneugenicity and clastogenicity/mutagenicity (RGO3, RGO8, and RGO6): the high surface oxidation of these samples might increase the presence of oxygen chemical groups on their surface, leading to a free radical's release which could eventually cause overwhelming ROS production and/or interaction with DNA.

3. Aneugenicity and no detected ROS production or mutagenicity/clastogenicity (GNP7, GNP8, silica): for this group, we can suppose that the mode of action does not involve oxidative stress but disturbance of mitotic apparatus (e.g., chromosome migration alteration during mitosis).

4. Increased ROS production, aneugenicity (GNP2, GNP3) and clastogenicity (GNP1): these samples show very small lateral size (approximately 1 µm or less) compared to the rest of the pool, which could allow the samples to directly interact with DNA. 5. Increased ROS production without any genotoxic effect (RGO5, RGO4, RGO7, RGO9, CB): these samples show an increase of ROS production which, at the tested exposure doses, might be controlled by the cell antioxidant defenses and will not cause genotoxicity.

Conclusion

This work allows having a better insight of the potential genotoxicity of GBMs and underlines that within the same family of nanomaterials (GBMs) and among only two subfamilies (GNPs and RGOs), we can still observe different outcomes and potential genotoxicity mechanisms.

We can conclude that most of the tested samples appear to induce an indirect, oxidative stressdriven genotoxicity. Using only two in vitro assays (HTS assay for genotoxicity and DCFH-DA assay for ROS production), combined with a relevant physicochemical characterization, we were able to formulate several hypotheses that shall be explored in the future in order to have a clearer insight of potential GBMs genotoxicity mechanisms. For a screening step and/or grouping purposes, this combination of DDR and DCFH-DA assays appears relevant and adapted to 2D carbonaceous materials such as graphene-based materials.

Considering the increasing demand for nanomaterials-based innovation and the necessity to test every nanoform of each nanomaterial, a systematic case-by-case approach appears impossible for toxicity study. Yet, the hazard assessment is a non-negotiable step for the entry in any market and an inescapable stage of research and development in industry. High throughput screening present undisputable advantages: it is quick, often standardized, and cheapest than considering a complete study of each nanomaterial and can allow to rank materials of interest before going any further in the development process. The approach proposed in this paper allows to have an insight on GBM's potential structure-activity relationship involved in genotoxicity and to have a clearer understanding of their mode of action. These indications can represent one more step toward safe-by-design GBMs. ICP results (ppm) GNP1 96,8 3,2 0,0 0,0 0,0 GNP2 93,7 6,3 0,0 0,0 0,0 GNP3 92,4 7,6 0,0 0,0 0,0 GNP7 95,4 4,2 0,0 0,4 0,0 GNP8 93,2 5,9 0,5 0,4 0,0 GNP9 93,2 5,7 0,7 0,3 0,0 rGO3 88,1 11,9 0,0 0,0 0,0 rGO4 91,7 7,2 0,0 1,0 0,0 rGO5 96,6 2,7 0,6 0,2 0,0 rGO6 84,1 15,9 0,0 0,0 0,0 rGO7 92,6 6,7 0,0 0,7 0,0 rGO8 82,8 17,2 0,0 0,0 0,0 rGO9 95,2 2,6 2,2 0,0 0,0 Amorphous Silica 96,9 2,6 0,0 0,5 0,0 CB1 97,2 2,3 0,0 0,5 0,0

XPS (atomic %)

C O N S Si

  serum and bovine pituitary extract (BPE)-free cell culture medium. The complete culture Pneumacult Ex-medium contains PneumaCult Ex Basal medium (Stemcell) added by PneumaCult Ex-50X supplement (Stemcell) and hydrocortisone (Stemcell) following the manufacturer's recommendations, antibiotics streptomycin (Streptomycin sulfate salt, Merck) and penicillin (Penicillin G sodium salt, Merck) respectively at 200 UI/mL and 50 µg/mL.

Figure 1 -

 1 Figure 2

Figure 2 -

 2 Figure 2 -Surface oxidation depending on the genotoxicity results.

Figure 3 -

 3 Figure 3 -Genotoxicity mode of action and ROS production.

Figure 4 -

 4 Figure 4 -Hypothetical genotoxicity mode of action of the tested GBMs.

Table 1 -

 1 Physicochemical features of the tested nanomaterials. BET: Brunauer, Emmett and Teller, EM: Electron Microscopy, XPS: X-ray spectroscopy.

		Specific surface	Mean lateral Size	Surface oxidation
		area, m²/g (BET)	in µm (EM)	atomic % (XPS)
	GNP1	283	1.3	3.2
	GNP2	439	0.7	6.3
	GNP3	692	0.5	7.6
	GNP7	89	10.9	4.2
	GNP8	168	17.3	5.9
	GNP9	119	38.6	5.7
	rGO3	545	8.3	11.9
	rGO4	880	31.6	7.2
	rGO5	830	7.0	2.7
	rGO6	270	32.0	15.9
	rGO7	810	15.1	6.7
	rGO8	440	1.0	17.2
	rGO9	870	1.1	2.6
	CB	112	0.4	2.6
	Amorphous Silica	160	0.1	70.0

Table 2 -

 2 DDR results.

	P53
	activation

Table 3 -

 3 Presentation of papers studying GBMs' genotoxicity. Studies colored in green: did not show genotoxicity, in yellow: showed mixed result, in red: showed genotoxicity. GO: Graphene Oxide, RGO: Reduced graphene oxide, S: Significant, NS: Non-significant.

	First Author Year	Number of GBM	GBM type	Physicochemical characteristics	Exposure time	Dose (µg/mL)	Model	Assay	Conclusions	Note
	Akhavan O 2012	4	RGO	Lateral Size : 11-3100 nm Thickness : 0.7-2.3nm Oxidation : 30-50%	1h	0.01-100 hMSCs	Comet assay Chromosomal aberration	S from dose 0.1 to 100 µg/mL S from dose 0.1 for some, some NS.	Obvious genotoxicity. The largest RGOs are less genotoxic
	Arbo MD 2019	1	GO	Lateral Size : ~550 nm Thickness : ~1nm	24h	10-100	H9c2	Comet assay Low molecular Weigh DNA fraction	S from dose 40 µg/mL	GO show moderate genotoxicity
	Bengtson S 2016	3	GO RGO	Size dispersion : 157-335 (DLS) Oxidation : 8-27%	24h	50-200	FE1	Comet assay	NS for all doses	No genotoxicity demonstrated.
					Lateral Size : 29-1000						Only the largest, less
	Jia PP	2019	6	G GO	nm Thickness : 1nm	24h	5-100	HEK 293T	Comet assay	S from dose 5 to 25µg/mL	oxidized G show genotoxicity at higher
					Oxidation : 5-33%						doses
	Li Y	2013	5	GO GO PEG GO BSA RGO PEG RGO BSA	Lateral Size : 30-450 nm Thickness : 1-15 nm	24h 48h 72h	200	U937	Comet assay	S at 24h for only GO S at 48h,for GO and RGO BSA S at 72h, for GO, RGO BSA, RGO PEG and GO BSA	A very high dose and long exposure are necessary to observe genotoxicity
	Mittal S	2016	3	GO, RGO Lateral Size : 90-750 nm	3h 6h	1-100	BEAS-2B A549	Micronucleus	S for dose 25 (3h) and 10 µg/mL(6h) for GO S from dose 1µg/mL for one RGO S at dose 100µg/mL (6h) for other RGO	Strong genotoxicity for 2 GBMs, none for one of them. The genotoxicity is lower when tested on A459
	Hashemi E 2016	2	GO RGO	Thickness : 0.8 nm	24h	0.1-400	SSCs	Comet	S from dose 10µg/mL (GO) and increased (no significativity found?) from dose 100µg/mL (RGO)	Strong genotoxicity for GO, moderate for RGO
										RNA efflux : S at dose 100µg/mL and
					Lateral Size : 11-3100	1			Comet assay	24h (RGOS) and S at dose 100µg/mL	Strong genotoxicity for
	Akhavan O 2012	2	RGOSs RGONRs	nm Thickness : 1 nm	5 24	0.01-100 hMSCs	Chromosomal aberration	and 1h (RGONR). Chromosomal abberation/Comet : S	RGONR (from lower dose and exposure
					Oxidation : 30-50%	96			RNA efflux	from dose 1µg/mL at 1 h exposure	time)
										(RGONR tested only)

Funding

This work was supported by Michelin.

Declaration of interest

Salma Achawi and Bruno Feneon are employees of Michelin company, a global tire and rubber goods manufacturer. Wang, A., Pu, K., Dong, B., Liu, Y., Zhang, L., Zhang, Z., Duan, W., and Zhu, Y., 2013. Role of surface charge and oxidative stress in cytotoxicity and genotoxicity of graphene oxide towards human lung fibroblast cells. Journal of applied toxicology: JAT, 33 (10), 1156-1164. Wang, K., Ruan, J., Song, H., Zhang, J., Wo, Y., Guo, S., and Cui, D., 2010

Supplementary data 2: GBMs internalization in BEAS-2B

To explore the internalization of GBMs in BEAS-2B cells, we made few acquisitions in optical and confocal microscopy.

Cell cultures were exposed to RGO4 (without metabolic activation) for 24 hours at 33.33 µg/cm² in 6well plates Cell bind surface (Costar) maintained at 37°C in a humidified 5% CO2 atmosphere in complete Pneumacult Ex-medium. After the beginning of exposure, cell cultures were grown for a period sufficient to undergo one mitosis and allow chromosome damage or impairment of chromosome segregation to lead to the formation of micronuclei in interphase cells. Cells were harvested, submitted to a hypotonic shock with Pneumacult Ex-medium diluted at 25% with distilled water (Versylene Fresenius), and then fixed. Giemsa stained interphase cells were then analyzed microscopically (In Cell Analyzer 6000, GE Healthcare, 60X, channel brightfiels / Channel Cy5) for the presence of micronuclei among mononucleated cells.

We selected the RGO4, one of our largest samples (mean lateral size of 31.6 µm) to make these acquisitions. For confocal microscopy, the sample was divided in 13 focal planes of 2 µm each, as shown in Figure S1. Considering that the average size of BEAS-2B is 20 µm, this allowed us to have a complete overview of the cell.

On the first picture (Figure S2A), we can observe a BEAS-2B cell with a micronucleus and on the second picture (Figure S2B), a cell with what we assumed could be an internalized agglomerate of RGO4. We chose a focal plane approximately located in the middle of the cell and observed in Brightfield mode the cell with the micronucleus (Figure S2C) and the assumed GBM (Figure S2D). It is clear that the micronucleus is absorbing the light like the rest of the chromatin in the cell whereas the assumed GBMs remain dark. We can then confirm that we actually observe a micronucleus on Figure S2C and a GBM agglomerate on figure S2D. Lastly, we observed the two slides in confocal microscopy, on 5 different focal planes from 1 to 13 for the micronucleus sample (Figure S2E) and for the GBM sample (Figure S2F).

The micronucleus and the RGO4 agglomerate both seem to follow the same pattern through the focal planes. For that reason, it appears that the GBM is internalized in the BEAS-2B cell.