A previous study compared the *in vivo* behavior of PEGylated-MoS₂, WS₂, and TiS₂ nanosheets of similar size.³⁹⁵ The nanomaterials (in the range of 100 nm size) showed predominant accumulation in the reticulum endothelial system (RES) such as liver and spleen after intravenous injection (10 mg/kg). In contrast with WS₂ and TiS₂, MoS₂ showed biodegradation in the liver and almost complete excretion after one month through urine and feces possibly due to the oxidation of MoS₂ into water-soluble molybdate species like MoO₄.² This is in agreement with the aforementioned mouse study in which *in vivo* degradation of MoS₂ in the lungs was shown with partial excretion of nanoparticles by way of extracellular vesicles.⁵⁶ With the aim of developing a drug delivery system, the distribution of MoS₂ and PEG-MoS₂ showed predominant accumulation of both nanomaterials in RES with a long retention time of more than 30 days for MoS₂.³⁹⁶ PEG-MoS₂ presented instead a faster excretion rate and was not observed in lungs, kidneys, heart, and brain. In another study, *in vivo* biodistribution of very small WS₂ (with an average size of 37.5 nm) was monitored after intravascular injection in mice. WS₂ nanosheets rapidly (within 1 h) accumulated in the liver, followed by distribution in the spleen, lung, and kidney, within 3 h. WS₂ nanosheets were completely excreted after 3 days. Intraperitoneal injection was also investigated and showed similar distribution but with a longer residence time of WS₂ for more than 10 days, mainly retained in the liver.³⁹⁷ It was evidenced that WS₂ transformation is not complete and stops at the level of tungsten oxide, which is more biopersistent.³⁹⁵ It would, however, be preferable that the tungsten oxide eventually evolved into soluble nontoxic tungstate in an oxidative environment. To date, few *in vivo* studies of hBN have been performed (although some studies have addressed BN nanotubes).³⁹⁸,³⁹⁹ Despite earlier intravascular studies in mice showing radioisotope-labeled PEG-hBN (20 mg/kg) accumulating in the liver, lung, heart and spleen, with major toxic effects in the heart,⁴⁰⁰ boron nitride has been used in many topical products such as cosmetics and has been established to be nontoxic in amounts used even if inhaled.⁴⁰¹ A rat study using an extremely high intravenous single dose of 1600 µg/kg hBN showed significant accumulation and damage at 24 h in the liver, kidney, heart, spleen and pancreas. However, 800 µg/kg showed no detrimental effect in terms of inflammation and cytotoxicity.³⁹⁰

## BIOTRANSFORMATION OF 2D MATERIALS

Many studies attempted to decipher cell uptake of GBMs, mainly taking into account the following parameters that drive 2D material interactions with biological systems: lateral dimensions and surface properties (e.g., oxygen containing groups and surface functionalization), cell types studied (phagocytic, nonphagocytic, cancerous or healthy cells) and material biotransformation (such as the presence of a surface-adsorbed biocorona and/or dissolution of the materials in various compartments in the body).⁴⁰²,⁴⁰³

Cellular uptake of GBMs has been extensively studied using professional phagocytes (macrophages) as well as other cell types such as lung and gastrointestinal epithelial cells. Recent work has shown that GO, regardless of lateral dimensions, interacts significantly with the plasma membrane of a panel of cell lines (BEAS-2B, NIH/3T3, HaCaT, 293T).⁴⁰⁴ However, the subsequent uptake mechanism is dependent on the lateral dimensions of the material. It was shown that small GO was internalized mainly via micropinocytosis, while ultrasmall GO

was mainly internalized via clathrin- and caveolae-mediated endocytosis. Interestingly, a shift from macropinocytosis to clathrin-dependent endocytosis in the uptake of small GO was demonstrated after 24 h. Importantly, both small GO and ultrasmall GO ended up in lysosomal compartments after 48 h.⁴⁰⁴ Several other studies have also demonstrated that lateral dimensions of GBMs govern cell uptake. Professional phagocytic cells are capable of taking up GBMs with a range of lateral dimensions,⁴⁰⁵ whereas nonphagocytic cells either do not take up large materials (>1 mm) or internalize the materials by micropinocytosis.⁴⁰⁴,⁴⁰⁶ Notably, uptake of nanomaterials is also influenced by the presence (or absence) of a protein biocorona.

**The Biocorona of 2D Materials.** The biocorona refers to the coating of biomolecules (proteins, lipids, sugars, nucleic acids, and metabolites) on the surface of nanomaterials, endowing the nanomaterials with a biological "identity".⁴⁰⁷ The composition of the biocorona of 2D materials drives cellular interactions not least in the immune system.²¹⁰ The protein corona is the most widely studied biocorona, but other biomolecules including lipids are also present in various biofluids and have been identified in the biocorona of GBMs.⁴⁰⁸ Early work showed that the biocorona mitigated the cytotoxicity of GO.⁴⁰⁹ Some investigators suggested that this is due to less uptake (endocytosis) of GBMs.⁴¹⁰ However, the latter studies were performed using a lung adenocarcinoma (nonphagocytic) cell line. Professional phagocytic cells may respond differently as the presence of specific proteins or "opsonins" in the corona may direct the nanomaterials toward specific uptake pathways. Moreover, surface modification, e.g., PEGylation, also modifies cellular uptake of GBMs.⁴¹¹ Different surface chemistries (e.g., GO, rGO, and FLG) and lateral dimensions also result in the adsorption of different proteins on the GBM surface⁴¹² but also in different relative orientations of these biomolecules,⁴¹³ exposing different epitopes for the interaction with cellular receptors. The percentages of immune-relevant corona proteins in graphene, borophene, and phosphorene were reported to be 41.3%, 46.5%, and 75.6%, respectively, indicating that graphene and borophene were not effective immune regulators. Several studies on protein corona on GBMs are available focusing on GO and how the protein layer mitigates its adverse effects in vitro.⁴¹⁴⁻⁴¹⁶ Theoretical studies suggested that the protein corona is able to reduce toxicity by reducing the physical interactions between the GO sheets and the cell membrane.⁴¹⁷ The role of the corona was also investigated in relation to GO cytocompatibility and antimicrobial properties.⁴¹⁸,⁴¹⁹ Other studies addressed the role of the biocorona for the uptake of FLG and rGO. Hence, long-term colloidal stable dispersions of FLG (lateral dimensions of about 200–300 nm) were prepared in the biological exposure medium in which the materials were studied (human serum, or fetal bovine serum, FBS) at the concentrations of 10%, 50%, and 100% in PBS. Naturally, the resulting FLG presented a biocorona of serum proteins.⁴²⁰ The authors were able to map some functionally relevant epitopes that are known to mediate binding to specific receptors on cells in the liver. In a follow-up study, the authors focused on one such corona protein, apolipoprotein A-I, and observed that the uptake of FLG was "somewhat" increased in cells expressing the cognate receptor, scavenger receptor B1.⁴²¹ Other investigators used sodium cholate to prepare stable aqueous dispersions of rGO. The authors hypothesized that changing the cholate concentration in the dispersion would
alter the surface properties of rGO.⁴²² To this end, rGO with varying concentrations of sodium cholate were prepared with or without a protein corona derived from a 1-h incubation in culture medium containing 10% FBS. The results revealed that the rGO dispersed in a lower surfactant concentration exhibited higher protein adsorption, and a stronger cytotoxicity. However, the surfactant itself also displayed cytotoxicity, and cell type-specific differences in susceptibility were noted. Thus, the interplay between rGO, the dispersant, and the biocorona, is complex. Finally, emerging data point toward the possibility of intracellular protein corona formation in macrophages.²³⁵

Other forms of biotransformation of GBMs apart from biocorona formation may also occur. Dissolution is an important parameter to take into account when addressing the biological or environmental effects of 2D materials "beyond graphene".⁹³ Biotransformation could be affected by the adsorption of biomolecules found in body fluids (for instance, in the airways, in the bloodstream, or in the gut), or be driven by enzymatic degradation in the body (for instance, following oral ingestion).³⁴⁴ In a recent study, an *in vitro* model of the intestinal epithelium was applied to simulate oral, gastric, and small intestinal digestion of GBMs.³⁴⁵ The authors concluded that occasional ingestion of small quantities of 2D materials such as hBN, MoS₂, WS₂, and GO is unlikely to be highly cytotoxic. The study reported significant agglomeration of all materials during digestion, especially GO, which was probably due to interactions with digestive proteins. Notably, the MoS₂ sheets had dissolved by ~75% after simulated digestion.³⁴⁵ Biologically relevant biotransformation of MoS₂ nanodots was reported in an *in vivo* study in which the MoS₂-HSA complexes were administered intravenously in mice at 5 mg/kg of body weight.³¹⁰ The nanodots were found to accumulate largely in the liver and spleen, and this biodistribution was accounted for by the presence of apoE in the biocorona. Moreover, biotransformation in the liver, potentially through the actions of phase I enzymes such as cytochrome P450 enzymes, resulted in the incorporation of molybdenum into molybdenum-dependent enzymes.

The biological interactions of MoS₂ are increasingly being studied.⁴²³ Early work demonstrated that exfoliated pristine and covalently functionalized MoS₂ were internalized while exerting minimal pro-inflammatory cytokine release in macrophages.¹⁵¹ MoS₂ is taken up by macrophages,²¹⁵ and the pro-inflammatory effects have been attributed to the adsorption of fibrinogen and IgG.²⁴⁶ MoS₂ nanosheets were found to interact with the plasma membrane of cells and are taken up via endocytosis in a size and cell type-dependent manner.⁴²⁴,⁴²⁵ Subcellular trafficking takes place through vesicular maturation from early, through late endosomes, toward the lysosomes, similarly to what has been reported for GO. Once reaching the lysosomes, the nanosheets either remain there or are slowly exocytosed from the cells.⁴²⁵ Larger MoS₂ nanosheets (average size: 700 nm), are primarily internalized in cells through phagocytosis/micropinocytosis, while smaller MoS₂ nanosheets (<300 nm), enter the cells either through the caveolar or clathrin-mediated uptake pathways, comparable to GO.⁴²⁴,⁴²⁵

**Biodegradation of 2D Materials.** Previous work has shown that carbon-based nanomaterials including SWCNTs and MWCNTs as well as GO and FLG can undergo enzymatic biodegradation.²⁸,²¹⁰ In an early study on enzymatic degradation of carbon-based nanomaterials, the plant enzyme,

horseradish peroxidase (HRP), and low amounts of hydrogen peroxide were applied.⁴²⁶ Soon, GO was also shown to undergo HRP-dependent degradation in "test-tube" experiments.⁴²⁷ Importantly, several mammalian peroxidases (myeloperoxidase, MPO; eosinophil peroxidase, EPO; lactoperoxidase, LPO) were subsequently shown to "digest" carbon nanotubes.⁴²⁸⁻⁴³⁰ Notably, LPO-driven degradation occurred even in the presence of a biocorona of pulmonary surfactant proteins and lipids.⁴³⁰ However, MWCNTs are not as effectively degraded as their single-walled counterparts.⁴³¹ Early work showed that GO can also undergo biodegradation in the presence of purified human MPO⁴³² and when incubated with activated human neutrophils.²³⁰ GO is also degraded to some extent by EPO,⁴³³ and GQDs have been shown to be degraded both by MPO and EPO.⁴³⁴ Studies performed in the Graphene Flagship also revealed that FLG can undergo neutrophil-driven degradation *ex vivo*, but the process was much slower than for GO (i.e., several days, as opposed to a couple of hours for GO).⁴³⁵

Overall, most degradation studies have used qualitative measurements to evaluate degradation, e.g., transmission electron microscopy, atomic force microscopy, and Raman spectroscopy, while few studies have explored quantitative approaches.⁴³⁶ However, it is noted that MPO is more effective than the HRP/H₂O₂ system.⁴³⁷

The degradation of GBMs has predominantly been studied in macrophages and neutrophils (and in the environment; see section Fungal and Bacterial Degradation). The mechanism of biodegradation in each cell type is distinct. Thus, while macrophage degradation of GBMs (such as graphdiyne oxide) was shown to occur intracellularly through a peroxynitrite-dependent pathway,²³⁴ neutrophils "digest" GBMs extracellularly, either through degranulation with the release of MPO, or through the formation of NETs that contain MPO as well as other granule proteins.²³⁰,⁴³⁸ Functionalization of GO with fMLP, a chemotactic peptide, was shown to trigger degranulation, leading to degradation in the absence of other stimuli.²⁷ Evidence of degradation of GO was also observed in the lungs of mice following pulmonary exposure; hence, Raman imaging revealed the progressive biotransformation of GO into less graphitic structures.²⁶⁶ However, the mechanism was not disclosed. Moreover, in a thorough evaluation of the *in vivo* fate of GO following intravenous injection, Newman et al.²⁹⁸ could show that GO present in spleen-resident macrophages gradually underwent biodegradation over a period of 9 months postexposure. This work offers important information on biological processing and degradation of GO in mammalian tissues.

Biodegradation of MoS₂ using HRP and MPO was found to be incomplete compared to that of GBMs.¹⁵¹ The degradation of MoS₂ in human THP-1 cells was monitored 24 h after exposure,⁴³⁹ although the mechanism of degradation was not disclosed. The distribution and translocation of polyvinylpyrrolidone (PVP)-modified MoS₂ nanosheets in cells, and their degradation in different biological environments (e.g., H₂O₂ alone, MPO plus H₂O₂, and catalase plus H₂O₂) have also been evaluated.⁴⁴⁰ It was found that MoS₂ nanosheets were completely decomposed when incubated with MPO in the presence of H₂O₂. Furthermore, it was found that intravenously administered PVP-MoS₂ was gradually cleared from mouse liver and spleen within 30 days. Early work on biodegradability of hBN evidenced the different actions of HRP and MPO. Partial oxidation was found using MPO, while
<figure><img src="image_35.png" /><figcaption>Figure 21. Hazard assessment of thermoplastic composites reinforced with reduced graphene oxide. (A) Characterization of rGO and abraded particles from PA6-rGO composites. SEM images of rGO, abraded particles from PA6-rGO composite and abraded particles from neat PA6. Animals (n = 3) were exposed by oropharyngeal aspiration to abraded polymer (PA6, 15 µg), abraded composite (PA6-rGO, 15 µg; with 2.5% rGO, hence 0.375 µg of rGO in 15 µg of PA6-rGO), reduced graphene oxide (rGO, 0.3 µg or 15 µg; 2.5% of 15 µg equals to about 0.3 µg), or negative control (BSA 0.1% in water). (B) Representative images of hematoxylin and eosin (H&E)-stained lung sections from mice exposed to rGO and abraded composites, following 1, 7, and 28 days after oropharyngeal aspiration. Arrows indicate the formation of granulomas after treatment with rGO.<sup>280</sup> Reproduced with permission from Chortarea, S.; Kuru, O. C.; Netkueakul, W.; Pelin, M.; Keshavan, S.; Song, Z.; Ma, B.; Gomes, J.; Abalos, E. V.; Luna, L. A. V.; Loret, T.; Fordham, A.; Drummond, M.; Kontis, N.; Anagnostopoulos, G.; Paterakis, G.; Cataldi, P.; Tubaro, A.; Galiotis, C.; Kinloch, I.; et al. Hazard Assessment of Abraded Thermoplastic Composites Reinforced with Reduced Graphene Oxide. *J. Hazard. Mater.* 2022, 435, 129053. Copyright 2022, Elsevier.</figcaption></figure>

HRP was unable to transform hBN even after 60 days.<sup>441</sup> A degradation study of hBN in a lysosome mimicking solution showed boron release after 30 days.<sup>442</sup>

In synopsis, the fate of GBMs has been extensively investigated, and we now know that certain GBMs (especially GO) are biodegradable, and that the formation of a protein corona (or biocorona) may reduce toxicity, while other 2D materials have not been studied to the same extent.

## LIFE CYCLE PERSPECTIVE ON 2D MATERIALS

To fully understand the environmental impact of GBMs and other 2D materials, a life cycle assessment is needed that applies a “cradle-to-grave” perspective on the production, use, and disposal of these materials.<sup>12,443</sup> Additionally, hazard assessment of 2D materials should take into account not only the as-produced or pristine material but should also adopt a life

cycle perspective. For comparison, previous work addressed the toxicity of aerosols generated from sanding polymer-coated MWCNT-embedded composites to better mimic the hazard that may be encountered by workers or consumers.<sup>444</sup> The authors found no evidence of free nanotubes in the aerosols and concluded that while the number of workers and consumers increases along the life cycle, toxicity and/or potential for exposure to the as-produced material may, in fact, be reduced. Here, we focus on the release of 2D nanofillers from polymer matrixes during the life cycle of the composite and explores acute impact on human health. For as-produced GBMs, there is a broad but as yet inconclusive understanding regarding their human health and environmental impact.<sup>3</sup> In contrast, much less data is available on materials embedded in composites, and on 2D material release after abrasion, combustion, or weathering of products.
Synthetic polymers and plastics are a relatively modern category of functional materials. Possessing attractive properties such as low density or easy processability in contrast to their metallic counterparts,445 polystyrene (PS), polyvinyl chloride (PVC), and polyester resins have been produced in large quantities since the 1930s.446 The reinforcement of plastics with carbon or glass fibers447 has led to a huge diversity of composite materials using different polymers and fillers. The use of carbon-based nanomaterials as fillers is not limited to mechanical improvements, but also to induce electrical conductivity, antifouling properties, or to reduce flammability. Carbon-based as well as 2D nanofillers have become very popular since their addition at low amounts (1–5 wt %) have shown to drastically improve composite properties. In parallel with the broad use of nanofiller-reinforced polymers, the potential exposure to humans and environment is increasing and therefore careful risk and hazard assessment is needed to avoid social and economic drawbacks.126,448 Matrix degradation occurs mainly by mechanical forces, aggressive chemicals, or weathering (e.g., UV light and temperature) and can result in increased release of particles into the environment and therefore human exposure. Normal use involves forces much less than that during intentional drilling or sanding, which makes aerosol production less of an issue compared to occupational use. It is also important when analyzing toxicity to consider that nanoparticles on surfaces, as opposed to compounded in a matrix, are not directly comparable.449 Recent studies have shown that graphene-based polyester resin composites are released during machining and in weathered samples.450 Cell viability levels of around 60% in A549 human lung cancer cells were observed with high dose (100 ppm) graphene nanosheets and graphene composites. Both graphene and resin demonstrated low toxicity although the authors acknowledged the presence of ambient particles from the spraying process that comprised of various metals. In another study conducted in the Graphene Flagship, abrasion of 2.5 wt % rGO reinforced polyamide (PA6), a representative thermoplastic, yielded a greater amount and smaller size of released particles (average size 1.91 µm) for the PA6-rGO composite (average particle size 3.16 µm) (Figure 21).280

This is in contrast to sanded GBM-reinforced polyurethane, where fewer fragments were released than that of matrix alone and showed no changes in particle size and no release of free GBM compared to the neat polymer.451–453 The abraded FLG-reinforced epoxy polymer showed no significant shift in the particle size distribution or free graphene compared to pure epoxy materials.296,454,455 When combusted, GBM-reinforced poly(lactic) acid films showed increased flame-retardant properties, and unburned GBMs could be found in the ashes, in higher quantities compared to neat polymers.456 Combustion of FLG-reinforced epoxy increased thermal stability of the composite, with no changes in amounts of particles released.457,458 When exposed to weathering, the composite reacted in the same way as the matrix, with lower amounts of particles released when exposed to UV light alone, but the same amounts upon simulated rain. However, free graphene flakes were observed.452,453 Based on the relatively few studies we can nevertheless extrapolate that depending on the combination of the matrix and the amount of nanofiller, only a moderate to low release of GBMs can be expected. However, despite the small amounts of GBMs released after the different treatments, careful assessment of abraded material is still warranted. Few studies have addressed the released

materials with respect to toxicity. FLG-reinforced epoxy after abrasion or after combustion displayed no or only very moderate and transient cytotoxicity.454,457 A similar result was observed in the multilaboratory analysis of rGO-PA6 abraded particles.280 Only as-produced rGO at the high dose of 40 µg/mL triggered adverse effects, most notably in macrophages. Since inhalation of airborne materials is mainly occupational, the effects after 1, 7, and 28 days after single pulmonary exposure were evaluated in mice. In agreement with in vitro data, rGO-PA6 abraded particles induced only modest and transient pulmonary inflammation.280

To sum up, relatively low amounts of 1–5 wt % 2D nanofillers in composites significantly improved polymer properties. During use and end-of-life, release of 2D nanofillers after abrasion, weathering or combustion is negligible. Particle size distribution after various stresses did not change compared to neat polymers; however, depending on combination of polymer and nanofiller, there remains a possibility of free or protruding GBMs. On the basis of current knowledge, the use of 2D nanofillers in composites could be considered a benefit that outweighs the risk. At present, this type of analysis is lacking for other 2D materials beyond GBMs.

## OCCUPATIONAL EXPOSURE TO 2D MATERIALS

Human inhalation exposure to 2D materials is most likely to occur in the occupational environment, and is associated with various activities in material processing and handling. Worker exposure to GBMs and other 2D materials is related to the safety and emission control of processes and activities in the synthesis and manufacturing. Occupational exposure can be potentially significant in end-of-life scenarios, such as recycling and waste handling. Following our previous review,³ few papers have been published on occupational exposure studies on these materials. The focus has been on production,459,460 or research and development461,462 in the workplace covering early life-cycle stages of the material, therefore leaving a dearth of knowledge on the later stages of occupational exposure including waste handling and recycling.

The Organisation for Economic Co-operation and Development (OECD) (ENV/JM/MONO(2015)19)463 and European Committee for Standardisation (CEN) approach (EN-16966:2018)464 for NOAA (Nano-Objects and their Aggregates and Agglomerates) assessment provides a clear and reliable view on the exposure situation in different and often dynamic industrial and R&D/Pilot occupational exposure scenarios. The widely harmonized multimetric tiered approach for workplace exposure measurement strategy and methods thus provides guidance for three tiers of assessment. Tier 1 is initial assessment, where the potential for release and emission of nano-objects (including 2D materials) into the workplace air is determined. Relevant workplace, process and production activity information is gathered structurally, according to best practices in occupational hygiene. Together with detailed material information, the possibility of release of nanomaterial can be considered. If Tier 1 shows potential for nanomaterial exposure, evaluation should proceed to Tier 2. Moreover, control or risk banding tools can be used to examine exposure potential at work. Most of these tools already have a Tier 1-type approach with structured questions focused on determination of potential release or exposure. Some examples can be found in the ISO technical specification on nanotechnologies (ISO/TS 12901-2:2014). Tier 2 is aimed at obtaining an indication of exposure to nano-objects. In this basic assess-
ment, exposure is investigated using easy-to-use and portable
measurement equipment to detect airborne nanomaterial
(nanoparticles, aerosol) levels in real-time during process and
activities. The off-line sampling and analysis of workplace air to
characterize possible nanomaterials is combined with real-time
assessment with techniques such as electron microscopy. It is
noted, however, that there are currently no consensus methods
for off-line analyses of most nanomaterials.⁴⁶⁵ Tier 3 is the
expert assessment of personal exposure to airborne particles.
The aim is to comprehensively characterize exposure to
airborne particles in the breathing zone of the workers. This
requires state-of-the-art techniques and methods, and evalua-
tions should include considerations and comparisons to the
corresponding reference values currently available. The
appropriate measurement techniques should cover the largest
size-range of particles currently available (10 nm to 10 µm),
have a suitable time resolution to monitor sudden changes in
concentrations due to work activities, and include size-
integrated metrics, such as particle surface area, number
concentration and mass concentration. Typically, as airborne
particles, 2D materials can be both nano- and micron-size
NOAA, with high variability in, for example, morphology and
state of agglomeration. Thus, it remains challenging to
selectively identify and quantify these particles for exposure
assessment purposes when current state-of-the-art online
instruments are designed for theoretical spherical object
measurements. At present, discussions and considerations of
the most useful combination of measurement methods and
metrics combining online and off-line assessments to assess
potential health effects are still ongoing.⁴⁶⁶ Exposure situations
and exposure potential in the workplace can be grouped
according to the state of the material during a specific work
process. Harmful emissions are most probable in the dry state,
and less probable in the liquid or paste state, when aerosol
formation is not likely (CEN EN-17058).⁴⁶⁷ Currently, most
production and related handling phases are in the liquid/paste
states of the material, and thus related emissions and exposure
potentials remain low.¹⁸²,⁴⁵⁹,⁴⁶⁰ The final stages of synthesis/
production process, when the raw material, such as produced
GBMs or other 2D materials, is dried and packed for further
use, are the most critical points regarding worker exposure, in
addition to equipment maintenance and cleaning, where dry
material can be released accidentally. The exposure (Tier 1 and
Tier 2) levels in CVD graphene production were previously
analyzed in an R&D laboratory.⁴⁶¹ The findings could not
prove any graphene particle emission during any process.
However, the need for more detailed Tier 3 assessment was
emphasized to achieve a more comprehensive assessment. A
GO pilot production process was then studied by applying Tier
2 methodology.⁴⁶⁸ The implemented safety measures proved
efficient, and no exposure was detected in the process. More
recently, GNP and GO emissions and exposures were
investigated during downstream industrial handling, showing
that powder handling contributes to the highest particle
emission and exposures.⁴⁶⁶ Overall levels remained low but the
importance of a multimetric approach to study worker
exposure was again highlighted. In the Tier 3 study of
producing and processing FLG in a pilot production
laboratory,⁴⁵⁹ potential release of FLG could not be excluded,
especially in the final process stages or equipment cleaning.
Exposure was not clear based on particle measurements, but
the filter sample collected from worker breathing zone
unexpectedly detected FLG. The low exposure levels detected

in recent studies can be related to utilization of proper and
efficient technical control measures, and using closed or
segregated systems to prevent release of harmful emissions in
workplace air. Nevertheless, challenges remain with respect to
risk assessment and risk management of 2D materials. The
multimetric approach can give relevant and essential
information on exposure scenarios, but the lack of associated
occupational exposure limit (OEL) values and the ever-
expanding knowledge gap on human health effects of 2D
materials have led to the use of precautionary principles in
exposure control and risk management. One suggested
approach is the utilization of safe-by-design principles to
cover the whole life cycle of the product, from innovation,
development, and production to end-of-life.⁴⁶⁹

REGULATORY PERSPECTIVE ON 2D MATERIALS

The bulk of all studies related to the toxicity or ecotoxicity of
2D materials are performed using nonstandardized protocols,
and the aim is not always to support regulation; instead, the
focus is on achieving a mechanistic understanding of the
(potential) toxicity of 2D materials. However, standardized test
protocols are required to support the regulation of up-to-date
materials. The OECD has addressed Test Guidelines (TGs)
and Guidance Documents (GDs) to evaluate the safety of
chemicals for over 60 years, and because nanomaterials are
chemicals, they are also included. The OECD TGs build on
the need of Mutual Acceptance of Data (MAD), hence
facilitating regulatory acceptance of data and avoiding
experimental duplication, especially of in vivo (animal) testing.
In Europe, nanomaterials fall under the Registration,
Evaluation, Authorisation and Restriction of chemicals
(REACH) regulation, (EC 1907/2006).⁴⁷⁰ However, partic-
ular issues regarding nanosized materials have been identified,
which challenge some of the TGs and GDs developed for
conventional chemicals. To this aim, the Working Party on
Manufactured Nanomaterials (WPMN)⁴⁷¹ was established in
2006 and the Sponsorship Programme for the Testing of
Manufactured Nanomaterials (Testing Guideline Programme,
TGP)⁴⁷² was launched in 2007. The Testing Programme
verifies testing methods applied on nanomaterials by pooling
the expertise of OECD member countries, some nonmember
countries, and other stakeholders to fund safety testing of
specific nanomaterials. Initially the Testing Programme
focused on 11 materials of industrial relevance, namely cerium
oxide, fullerenes, dendrimers, gold nanoparticles, MWCNTs,
nanoclays, silicon dioxide, silver nanoparticles, SWCNTs,
titanium dioxide, and zinc oxide. Following on from these
initial activities, efforts focused on identification of needs
regarding adaptations of guidelines to nanomaterials (ENV/
JM/MONO(2009)21).⁴⁷³ These efforts fall under the OECD
WPMN in collaboration with the Working Group of National
Co-ordinators of the TGP (WNT), and the status of the work
is reviewed yearly through a work plan publication.

The large European research project NanoReg (85 partner institutes) funded under the FP7 program had as an overarching aim the development of a common European approach to the regulatory testing of nanomaterials in terms of environmental, health and safety issues. One of the key results was to evaluate the applicability of several TGs to nanomaterials and to highlight shortcomings where these were present. Several recommendations were made, which have been summarized in the so-called ProSafe “white paper”.⁴⁷⁴ To meet the regulatory needs, an action plan was set up in 2017 to
support the amendment and development of TGs for nanomaterials and especially nanoforms in REACH. This is known as the “Malta Initiative” (so named as the initiative arose during the Maltese EU Council presidency) and brings together EU member states, the European Commission (including Directorate-General Research and Innovation, Directorate-General Environment, and Joint Research Centre, JRC), the European Chemicals Agency (ECHA), industries, and other institutions. The Malta Initiative is a voluntary instrument without an official/legal mandate. The OECD MAD ensures that test results generated in accordance with OECD TGs and the OECD Principles of Good Laboratory Practice (GLP) are accepted in all OECD countries and adherent countries. Therefore, OECD TGs are key for internationally harmonized and standardized safety testing of chemicals and materials for governments, industry, and academia. To date, the Malta Initiative has facilitated updating and modification of 18 TGs, ensuring that they are fit for purpose for nanomaterials. The output of the Malta Initiative shows that a coordinated effort leads to successful TG development. Such effort includes: (a) funding of researchers for the validation and harmonization of test methods, and (b) an international platform for collaboration and exchange between stakeholders. The general aims are to identify relevant end points and methods ready for validation and harmonization; to support collaboration of researchers, regulators, and industry in TG development; to ensure the development of test methods that are operable and useful in (pre) regulatory and scientific testing; to increase the chances for (effective) adoption by OECD member countries. Despite the important progress made, gaps in method developments for nanomaterials remain, and up-to-date developments in material innovations require further method developments. In collaboration with the OECD, the European Commission has funded further activities under the Malta Initiative to foster development of up-to-date or adapted TGs or GDs for the safety assessment of nanomaterials. Thus, Horizon 2020 projects such as NanoHarmony and Gov4Nano, in collaboration with the NanoMet project at OECD, are focused on the development of TGs and GDs to cover regulatory gaps identified by the Nanomaterial Expert Group (NMEG) at ECHA.

Despite the large efforts put forward by different initiatives at the EU level, uncertainties regarding safety assessment of nanomaterials still exist. One example is the recent revoking of the Commission Delegated Regulation of 2019 regarding harmonization and labeling of TiO₂ as a carcinogenic substance by inhalation in certain powder forms by the European Court of Justice (EU Press Release No. 190/22).⁴⁷⁵ Thus, this points to a lack of reproducibility of results as a consequence of a lack of harmonized protocols and the long time required to achieve approval of protocols at the OECD level. To accelerate this process, several experts have already published adapted protocols to address particular issues related to nanomaterials, even if these have not yet obtained OECD approval. Examples include protocols produced in European Commission funded projects (NanoReg, NanoTest, PATROLS, GRACIOUS, RiskGONE, etc.) such as the adaptation of cytotoxicity tests to avoid nanomaterial interference,⁴⁷⁶ adaptations of genotoxicity assays so that they are more reliable for nanomaterials,⁴⁷⁷ and the implementation of F.A.I.R. (findability, accessibility, interoperability, and reusability) data principles in nanosafety research.⁴⁷⁸ However,

gaps still exist, and a prioritization scheme is required like the one put forward by the NMEG that led to the work performed in Gov4Nano and NanoHarmony. In a recent review coordinated by the Dutch National Institute for Public Health and the Environment (RIVM), a comprehensive survey was provided of the information requirements in all areas of European legislation that are applicable to nanomaterials and needs for further action to address nanospecific issues were identified.⁴⁷⁹ Overall, harmonization efforts concluding at OECD level will help improving transferability and reproducibility of results from different laboratories and, hence, will contribute to the current uncertainty gathered around nanosafety data from the literature, supporting regulatory decision making and making a positive impact in technology development. This is certainly true not only for “traditional” nanomaterials but for 2D materials as well. The Graphene Flagship has addressed the need for the harmonization of OECD TGs and GDs with respect to the ecotoxicity and human toxicity testing of GBMs. Regarding ecotoxicity, data generated in the framework of the Graphene Flagship may be useful in the revision of the GD on Aquatic Toxicity Testing of Nanomaterials, generating annexes to explain the applicability of TG 201 (Algae and Cyanobacteria Growth Inhibition Test), TG 202 (Daphnia Acute Immobilization Test), and TG 203 (Fish Acute Toxicity Test) to nanomaterials and GBMs. Additional work has been done in relation to a more recent GD on assessing the apparent accumulation potential for nanomaterials, and although all the protocols and steps for the application of TG 305 (Bioaccumulation in Fish) to GBMs are evident, the lack of routine methodologies for the determination of GBM concentrations in biological tissues constitutes an essential limitation.

Regarding OECD TGs related to human toxicity, the work performed in the Graphene Flagship has focused mainly on skin safety. In general, OECD TGs predicting skin toxicity assess skin irritation, corrosion, and sensitization. These *in vitro* TGs employing a 3D model of artificial epidermis (OECD TG 439 and 431) can be adopted for GBMs without modification,¹⁸⁸,¹⁹⁰ with OECD TG 442B also demonstrated to be applicable to GBMs.¹⁸⁷ However, technical limitations with regard to the TGs evaluating *in vitro* the initial three phases of skin sensitization AOPs, namely reactivity with peptides (OECD TG 442C), keratinocytes activation (OECD TG 442D), and dendritic cells activation (OECD TG 442E), were identified and relevant modifications of the procedures are needed before TG adoption for GBMs. Initial information has been included in an OECD report on the applicability of OECD TG 442D in nanomaterial testing.

Members of the Graphene Flagship have thus assessed skin irritation using the SkinEthic™ reconstructed human epidermis, following OECD TG 439. Even though not validated for nanomaterials, the OECD TG 439 turned out to be applicable also for GBM testing, since no interference with the methylthiazoloyldiphenyl-tetrazolium bromide reduction, used as a readout, was found.¹⁸⁸ On the same model, skin corrosion of GBMs was very recently evaluated following the OECD TG 431.¹⁹⁰ Furthermore, skin sensitization by FLG and GO was evaluated following the OECD TG 442B (Local Lymph Node Assay).¹⁸⁷ This *in vivo* study following OECD TG 442B demonstrated the absence of skin sensitization properties for two representative GBMs, FLG and GO.
CONCLUSIONS AND FUTURE PERSPECTIVES

The Graphene Flagship (2013–2023), a combined academic-industrial consortium funded by the European Commission, has succeeded in building a foundation for a graphene industry in Europe.<sup>480,481</sup> Human health and environmental issues have always been a part of this endeavor. Hence, we prepared and published a “midterm report” in 2018 with a survey of the literature on safety assessment of graphene and related materials.<sup>3</sup> Here, we have expanded this discussion to other 2D materials. Together, the two reports provide an overview of the state-of-the-art of the safety assessment of graphene and other 2D materials with respect to human health and the environment. We remarked in the previous review that all GBMs cannot be grouped together as one material. Indeed, GBMs may vary considerably in terms of intrinsic physicochemical properties,<sup>6</sup> leading to differences in terms of their interactions with biological systems. This is also true for the ever-expanding universe of other 2D materials including MXenes, TMDs, etc.<sup>482</sup> Since limited (eco) toxicological data are available on more recent advanced 2D materials like MXenes, 2D metal organic frameworks, perovskites, layered double hydroxides, and many others, efforts to assess their potential impact on health and the environment are warranted. Furthermore, as we have discussed in the present review, GBMs and other 2D materials may undergo biotransformation. It follows that it is not sufficient to characterize the properties of as-produced 2D materials; toxicologists must also consider the many transformations (e.g., agglomeration, dissolution/degradation, coronation) that may occur in the natural environment or in the human body, both in the extracellular and in the intracellular compartment. Thus, 2D materials may be regarded as dynamic entities, displaying an evolving synthetic identity as well as a biological identity that is determined, at least in part, by the adsorption of biomolecules. This is certainly exciting and worthy of exploration from an academic point of view, but the realization that 2D materials are not one single material, and the understanding that these materials interact in a dynamic fashion with biological systems, also has considerable implications for the regulation of 2D materials. Regulation could have positive and negative effects on the innovation process, but we ignore the safety assessment of 2D materials and other advanced materials at our peril.

Significant advances have been made in the development of 2D material-based sensors to detect biomarkers for various diseases. In certain cases, tissues from different organs might come into direct contact with the materials embedded into these devices. Although the surface contact of the organs to the 2D materials is likely very limited, we suggest the scientific community to carry out toxicity studies about the potential undesired effects on tissues beyond biomarker sensing.<sup>483,484</sup>

In the present review, we have also discussed “green” chemistry approaches to minimize the environmental footprint of 2D materials. This aligns well with the “safe-and-sustainable-by-design” (SSbD) concept that is embedded in the European Commission’s Chemicals Strategy for Sustainability (CSS). The so-called SSbD approach (OECD ENV/CBC/MONO(2022)30)<sup>485</sup> requires “life cycle thinking” to ensure sustainability along the entire value chain. Toxicological assessment is, of course, an important element, but chemicals and, by extension, nanomaterials, should be safe and sustainable both at the manufacturing phase, at the use phase, and at end-of-life of the product.

We have also attempted a careful description not only of the test material, but also the test system, and to the extent that this is possible, the test method/assay/end point. The reason for this is very simple: to enable a comparison between different studies. Indeed, the research community has recently addressed the need for a “Minimum Information Reporting in Bio-Nano Experimental Literature” (MIRIBEL) for published accounts of so-called bionano research.<sup>486,487</sup> This encourages researchers in the field of toxicology to adhere to certain reporting standards in order to enhance research quality and avoid unnecessary duplication of experimental work.

It is worth noting that the interactions between 2D materials and biological systems are reciprocal. Hence, 2D materials may have an impact on cells and tissues, causing toxicity, but biological systems can to a certain extent detoxify 2D materials through enzymatic degradation. Importantly, this has been demonstrated both in mammalian systems, and in the environment. Understanding and controlling these processes may suggest novel strategies by which to mitigate the toxicity of 2D materials. This is certainly relevant not only for inadvertent exposure to 2D materials, but may also have ramifications for the clinical translation of (selected) 2D materials.

Looking ahead, we expect to see increasing numbers of 2D materials and 2D material-based applications in the years to come, and safety assessment will thus remain a strong priority, and should be viewed as an integral component of every 2D material research or innovation project. To this end, we need to develop and deploy robust test protocols based on relevant model systems to ensure that emerging 2D materials are safe for human health and the environment.

AUTHOR INFORMATION

Corresponding Authors

Bengt Fadeel – Nanosafety & Nanomedicine Laboratory, Institute of Environmental Medicine, Karolinska Institutet, 177 77 Stockholm, Sweden; ● orcid.org/0000-0001-5559-8482; Email: bengt fadeel@ki.se

Alberto Bianco – CNRS, UPR3572, Immunology, Immunopathology and Therapeutic Chemistry, ISIS, University of Strasbourg, 67000 Strasbourg, France; ● orcid.org/0000-0002-1090-296X; Email: a.bianco@ibmc-cnrs.unistra.fr

Authors

Hazel Lin – CNRS, UPR3572, Immunology, Immunopathology and Therapeutic Chemistry, ISIS, University of Strasbourg, 67000 Strasbourg, France

Tina Buerki-Thurnherr – Empa, Swiss Federal Laboratories for Materials Science and Technology, Laboratory for Particles-Biology Interactions, 9014 St. Gallen, Switzerland; ● orcid.org/0000-0003-3723-6562

Jasreen Kaur – Nanosafety & Nanomedicine Laboratory, Institute of Environmental Medicine, Karolinska Institutet, 177 77 Stockholm, Sweden

Peter Wick – Empa, Swiss Federal Laboratories for Materials Science and Technology, Laboratory for Particles-Biology Interactions, 9014 St. Gallen, Switzerland; ● orcid.org/0000-0002-0079-4344

Marco Pelin – Department of Life Sciences, University of Trieste, 34127 Trieste, Italy; ● orcid.org/0000-0002-4306-7411
**Aurelia Tubaro** — Department of Life Sciences, University of Trieste, 34127 Trieste, Italy

**Fabio Candotto Carniel** — Department of Life Sciences, University of Trieste, 34127 Trieste, Italy; ● orcid.org/0000-0002-8277-4725

**Mauro Tretiach** — Department of Life Sciences, University of Trieste, 34127 Trieste, Italy

**Emmanuel Flahaut** — CIRIMAT, Université de Toulouse, CNRS, INPT, UPS, 31062 Toulouse, France; ● orcid.org/0000-0001-8344-6902

**Daniel Iglesias** — Facultad de Ciencias y Tecnologías Químicas, Universidad de Castilla-La Mancha (UCLM), 13071 Ciudad Real, Spain; Instituto Regional de Investigación Científica Aplicada (IRICA), Universidad de Castilla-La Mancha (UCLM), 13071 Ciudad Real, Spain

**Ester Vázquez** — Facultad de Ciencias y Tecnologías Químicas, Universidad de Castilla-La Mancha (UCLM), 13071 Ciudad Real, Spain; Instituto Regional de Investigación Científica Aplicada (IRICA), Universidad de Castilla-La Mancha (UCLM), 13071 Ciudad Real, Spain; ● orcid.org/0000-0003-3223-8024

**Giada Cellot** — International School for Advanced Studies (SISSA), 34136 Trieste, Italy; ● orcid.org/0000-0001-9198-8402

**Laura Ballerini** — International School for Advanced Studies (SISSA), 34136 Trieste, Italy; ● orcid.org/0000-0001-8420-0787

**Valentina Castagnola** — Center for Synaptic Neuroscience and Technology, Istituto Italiano di Tecnologia, 16132 Genova, Italy; IRCCS Ospedale Policlinico San Martino, 16132 Genova, Italy

**Fabio Benfenati** — Center for Synaptic Neuroscience and Technology, Istituto Italiano di Tecnologia, 16132 Genova, Italy; IRCCS Ospedale Policlinico San Martino, 16132 Genova, Italy; ● orcid.org/0000-0002-0653-8368

**Andrea Armirotti** — Analytical Chemistry Facility, Istituto Italiano di Tecnologia, 16133 Genoa, Italy; ● orcid.org/0000-0002-3766-8755

**Antoine Sallustrau** — Département Médicaments et Technologies pour la Santé (DMTS), Université Paris-Saclay, CEA, INRAE, Gif-sur-Yvette 91191, France

**Frédéric Taran** — Département Médicaments et Technologies pour la Santé (DMTS), Université Paris-Saclay, CEA, INRAE, Gif-sur-Yvette 91191, France; ● orcid.org/0000-0001-5461-329X

**Mathilde Keck** — Département Médicaments et Technologies pour la Santé (DMTS), Université Paris-Saclay, CEA, INRAE, Gif-sur-Yvette 91191, France; ● orcid.org/0000-0003-4717-3446

**Cyrill Bussy** — Nanomedicine Lab, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, National Graphene Institute, Manchester M13 9PT, United Kingdom; ● orcid.org/0000-0001-8870-443X

**Sandra Vranic** — Nanomedicine Lab, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, National Graphene Institute, Manchester M13 9PT, United Kingdom; ● orcid.org/0000-0002-6653-7156

**Kostas Kostarelos** — Nanomedicine Lab, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, National Graphene Institute, Manchester M13 9PT, United Kingdom; ● orcid.org/0000-0002-2224-6672

**Mona Connolly** — Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), E-28040 Madrid, Spain

**José Maria Navas** — Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), E-28040 Madrid, Spain

**Florence Mouchet** — Laboratoire Ecologie Fonctionnelle et Environnement, Université de Toulouse, CNRS, INPT, UPS, 31000 Toulouse, France

**Laury Gauthier** — Laboratoire Ecologie Fonctionnelle et Environnement, Université de Toulouse, CNRS, INPT, UPS, 31000 Toulouse, France

**James Baker** — TEMAS Solutions GmbH, 5212 Hausen, Switzerland

**Blanca Suarez-Merino** — TEMAS Solutions GmbH, 5212 Hausen, Switzerland

**Tomi Kanerva** — Finnish Institute of Occupational Health, 00250 Helsinki, Finland

**Maurizio Prato** — Center for Cooperative Research in Biomaterials (CIC biomaGUNE), Basque Research and Technology Alliance (BRTA), 20014 Donostia-San Sebastián, Spain; Ikerbasque, Basque Foundation for Science, 48013 Bilbao, Spain; Department of Chemical and Pharmaceutical Sciences, University of Trieste, 34127 Trieste, Italy; ● orcid.org/0000-0002-8869-8612

Complete contact information is available at: https://pubs.acs.org/10.1021/acsnano.3c09699

### Notes

The authors declare no competing financial interest.

### ACKNOWLEDGMENTS

The authors are supported by the European Commission through the Graphene Flagship Project (grant agreement no. 881603) (work package on Health & Environment, and SafeGraph SH11 spearhead project), and the Centre National de la Recherche Scientifique (CNRS) (A.B., E.F.), the Interdisciplinary Thematic Institute SysChem, through the IdEx Unistra (ANR-10-IDEX-0002) within the French Investments for the Future Program (A.B.), by the Commissariat à l'Energie Atomique et aux Energies Alternatives (A.S., F.T., M.K.), and from the Swiss National Science Foundation (grant no. 310030_169207) (T.B., P.W.). M.C. had a contract in the framework of the project 2018-T2/AMB-11392 granted and funded by the Community of Madrid. This study forms part of the Advanced Materials program and was supported by MCIN (project PID2020-113080RB-I00) with funding from European Union NextGenerationEU (PRTR-C17.11) and by the Junta de Comunidades de Castilla-La Mancha (SBPLY/21/180501/000176) (E.V.). Parts of some figures were created with BioRender.com.

### VOCABULARY

**Two-dimensional materials**, a family of ultrathin crystalline materials wherein atoms are organized in single- or few-layers, for instance graphene (a crystalline allotrope of carbon); **Green chemistry**, chemistry focusing on environmental impacts with the design of chemical products and use of processes that reduce the use or generation of hazardous substances; **Biocorona formation**, the coating of biomolecules
(proteins, lipids, sugars, nucleic acids, etc.) onto the surface of materials, endowing the materials with a different biological "identity"; **Biodegradability**, the ability of a material to decompose into smaller fragments and/or ions after interactions with oxidative enzymes present in the human body or in the environment; **Biodistribution**, tracking where compounds of interest travel in an experimental animal or human subject the biodistribution of materials is controlled by many factors including size; **Life cycle assessment**, methodology for the assessment of environmental impacts associated with all stages of the life cycle of a product, process, or service (i.e., from "cradle" to "grave"); **Safe and sustainable-by-design**, an approach to support design, development, production and use of chemicals and materials while minimizing impacts on health and the environment

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