What are the Conclusion of the Short-Term Inhalation Study of Graphene Oxide Nanoplates


Nano

Graphene oxides possess unique physicochemical properties with important potential applications in electronics, pharmaceuticals and medicine.

Inhalation Toxicity of Graphene Oxide

However, the toxicity after inhalation exposure to graphene oxide has not yet been elucidated. Therefore, in this study, a short-term analysis of the inhalation toxicity of graphene oxide was performed using a nose-only inhalation system and male Sprague-Dawley rats.

A total of four groups (15 rats per group) were exposed to: (1) control (fresh air), (2) low concentration (0.76 ± 0.16 mg/m3), (3) moderate concentration (2.60 ± 0.19 mg/m3), and (4) high concentration (9.78 ± 0.29 mg/m3). Rats were exposed to graphene oxide for 6 h/day for 5 days, followed by recovery periods of 1, 3, and 21 days.

No significant changes in body or organ weights were observed after short-term exposure or during the recovery period. Similarly, no significant systemic effects of toxicological significance were detected in hematologic assays, inflammatory markers in bronchoalveolar lavage (BAL) fluid, cytokines in BAL fluid, or blood biochemical assays after graphene oxide exposure or during the post-exposure observation period.

In addition, no significant differences were observed in the differentiation of BAL cells, such as lymphocytes, macrophages, or polymorphonuclear cells. Graphene oxide-loaded alveolar macrophages as a spontaneous clearance response were observed in the lungs of all concentration groups from 1 day to 21 days after exposure.

Histopathologic examination of the liver and kidneys revealed no significant histopathologic lesions relevant to the test article. Importantly, similar to previous reports on graphene inhalation, only minimal or imperceptible toxicity of graphene oxide was observed in the lungs and other organs in this short-term inhalation study conducted only via the nose.

Introduction

Graphene is defined as a single layer of carbon atoms, with each atom connected to three neighbors in a honeycomb structure (ISO TS 80004-3, 2010). In the case of graphene oxide (GO), the epoxide (1,2-ether) and hydroxyl functional groups are covalently bonded on each side of the basal plane, while the carboxyl groups are located at the edges (Balapanuru et al., 2010).

This strong and lightweight nanomaterial is expected to be used in many industrial fields, including biomedical applications, electronics, energy, and sensors, but this also raises concerns about human exposure and environmental and occupational hazards (Sanchez et al., 2012; Yang et al., 2015; Lee et al., 2016; Park et al., 2017).

Occupational exposure to graphene nanomaterials may occur in air during the production of graphene nanomaterials through oxidation and reduction processes, and the exposed forms may include individual graphene nanoplatelets, aggregates, or agglomerates (Lee et al., 2016).

Graphene oxide (200-500 nm lateral size) has previously been found to induce cytotoxicity and genotoxicity in human lung fibroblasts in a dose-dependent manner (1-100 μm/ml), with oxidative stress and surface charge of graphene oxide shown to mediate toxicity (Wang et al., 2013).

At the cellular level, graphene oxide (160 – 780 nm) did not penetrate A549 cells, but caused oxidative stress in a dose-dependent manner and resulted in a slight loss of cell viability only at high concentrations, suggesting that graphene oxide is a relatively safe material (Chang et al., 2011).

Furthermore, graphene oxide injections into the tail veins of rats for 7 days at concentrations of 2.5, 5, and 10 mg/kg body weight did not cause behavioral changes in field experiments and functional observational battery tests, but caused inflammation of the lungs, liver, and spleen at a high concentration (10 mg/kg). Graphene oxide injections also decreased cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein levels (Li et al., 2016).

Another study on the acute inhalation toxicity of graphene oxide also showed that acute inhalation exposure to graphene oxide at concentrations of 0.46 and 3.76 mg/m3 induced minimal toxic responses in the lungs of male Sprague-Dawley rats without increasing inflammatory markers (Han et al., 2015).

Regardless, the surface reactivity, size, and dispersion status of graphene nanomaterials play important roles in inducing toxicity and biodistribution of graphene nanomaterials.

In addition, oxidative stress and induction of inflammatory response are also important for the induction of toxicity related to the biodistribution of graphene nanomaterials (Ema et al., 2017).

Therefore, despite several studies on the acute inhalation toxicity of graphene oxide, there has been no study on the repeated inhalation toxicity of graphene oxide, which is more relevant for evaluating the hazards of repeated exposure to graphene nanomaterials in the workplace.

Accordingly, this study collected the necessary data for a safety assessment of graphene oxide, including pulmonary toxicity, systemic toxicity via hematology, blood biochemistry, and histopathology in major organs after 5-day exposure and after various post-exposure periods.

Materials and Methods

Characterization of Graphene Oxide

The graphene oxide nanopowder (GO-A200, IGH20160414; thickness 1~2 atomic layer) used in the experiments was provided by Grapheneall Co. (Gwinsean-gu, Suwon-si, Gyeonggi-do, South Korea). The physicochemical properties of the graphene oxide nanopowder were characterized, including its elemental contents, lateral size, thickness, D/G ratio, crystallinity, and conductivity.

The elemental contents were determined by thermogravimetric analysis (TG/DTA 7300, Seiko Inc., Chiba, Japan) and inductively coupled plasma mass spectrometer (ICP-MS, Agilent Technologies 7300, Santa Clara, CA) analysis, D/G ratio determined by Raman spectroscopy (WITec alpha 300, Ulm, Germany), structural analysis was performed with a Rigaku Ultima IV X-ray diffractometer (Tokyo, Japan), zeta potential measured with a Malvern ZS90, He-Ne 633 laser (UK) ,the viscosity measured with a viscometer (PCE RVI 6, Southampton Hampshire, UK), the surface area measured with a BELSOPR-min II (MicrotracBEL, Osaka, Japan) using the Brunauer-Emmett-Teller method, and the thickness of the graphene layers analyzed with atomic force microscopy (AFM, Park System NX 10, Seoul, Korea).

The graphene oxide aerosols were collected on a TEM grid (copper grid, Formvar/Carbon 200 mesh, TEDpella, CA) and analyzed using a field emission transmission electron microscope (FE-TEM, JEM2100F, JEOL, Tokyo, Japan) with an EDX (EDX, TM200, Oxford Instruments plc, Oxfordshire, UK) at an accelerating voltage of 200 kV (NIOSH 1994). and the thickness of the graphene layers analyzed with atomic force microscopy (AFM, Park System NX 10, Seoul, Korea). The graphene oxide aerosols were collected on a TEM grid (copper grid, Formvar/Carbon 200 mesh, TEDpella, CA) and analyzed using a field emission transmission electron microscope (FE-TEM, JEM2100F, JEOL, Tokyo, Japan) with an EDX (EDX, TM200, Oxford Instruments plc, Oxfordshire, UK) at an accelerating voltage of 200 kV (NIOSH 1994). and the thickness of the graphene layers analyzed with atomic force microscopy (AFM, Park System NX 10, Seoul, Korea).

The graphene oxide aerosols were collected on a TEM grid (copper grid, Formvar/Carbon 200 mesh, TEDpella, CA) and analyzed using a field emission transmission electron microscope (FE-TEM, JEM2100F, JEOL, Tokyo, Japan) with an EDX (EDX, TM200, Oxford Instruments plc, Oxfordshire, UK) at an accelerating voltage of 200 kV (NIOSH 1994). UK) at an accelerating voltage of 200 k V (NIOSH 1994).UK) at an accelerating voltage of 200 k V (NIOSH 1994).

Aerosol Generation

Male Sprague-Dawley (SD) rats were exposed to graphene oxide nanopowder for 5 days using a Nase-only exposure system (HCT, Icheon, Korea). The graphene oxide nanopowder was generated using an atomizer (AG-01, HCT, Icheon, Korea) ( Table S1) with purified air as the carrier gas.

Fresh air was used as a control, while different water suspensions were used to generate different aerosols: 0.04 mg/ml for low concentration, 0.19 mg/ml for medium concentration, and 0.77 mg/ml for high concentration. Airflow was maintained at 30 liters per minute (l/min) using a mass flow controller (MFX, FX-7810CD-4V, AERA, Tokyo, Japan), and the flow rate to each nostril was 1 l/min. The AC supply was maintained at 99.56 ± 0.07 V (mean ± SE). The target concentrations of graphene oxide nanopowder were 0.625, 2.5, and 10 mg/m 3for the low, medium, and high concentrations, respectively.

Mass median aerodynamic diameter (MMAD) was measured using a MOUDI 125NR (cascade impactor, MSP Co, Shoreview, MN) at a flow rate of 10 l/min. An aluminum foil filter coated with grease was used in each stage to minimize particle bounce. The aerosol mass collected on the filters was determined as the difference between the post and pre-weights of the filters. The geometric standard deviation (GSD) of the distribution was derived from the cumulative mass distribution of the filters.

Monitoring of graphene oxide aerosol in the inhalation chamber

Particle size distribution was measured using a dust monitor (model 1.1.09, Grimm Technologies Inc. Douglasville, GA) and a scanning nanoparticle sizer (SMPS, HCT Co., Ltd., Icheon, Korea). The mass concentration of graphene oxide was determined gravimetrically (as post-weight minus pre-weight) by sampling on a PVC filter (polyvinyl chloride, size: 37 mm and pore size 5.0 µm) at a flow rate of 1.0 L/min.

Elemental Carbon Analysis

To quantify the elemental carbon (EC) content in the graphene oxide aerosols, quartz filters (37 mm diameter quartz fiber filters, SKC Inc., Eighty-Four, PA, USA) were also used to collect the total suspended particles (TSP) and analyze the EC concentration. The quartz filters were then analyzed to determine the mass concentration of EC in the air.

The filters were analyzed according to NIOSH Manual of Analytical Method (NMAM) Method 5040 ( NIOSH, 2003 ) , which is currently recommended by NIOSH to assess exposure to CNTs and carbon nanofibers (CNFs) ( NIOSH, 2013). An organic carbon (OC) analysis is also routinely used to characterize carbonaceous nanomaterials as well as carbonaceous impurities in engineered nanomaterials (ENMs). In this study, the quantification reporting limit (LOQ) for EC, organic carbon, and total carbon was 2 μg, 2 μg, and 4 μg/filter, respectively, and the limit of detection (LOD) was 0.6 μg/filter for each analyte category.

Animals and Conditions

Six-week-old male specific pathogen-free SD rats were obtained from OrientBio (Seongnam, Korea) and acclimatized for two weeks before inhalation exposure was initiated. During acclimatization and inhalation exposure, rats were maintained in a controlled temperature (22 ± 0.83 °C) and humidified (47 ± 0.69%) environment with a 12-h light-dark cycle. The rats were fed a rodent chow (Woojung BSC, Suwon, Korea) and filtered water ad libitum. During the acclimation period, the animals were trained for 6 h/day to adapt to the nose-only inhalation chamber.

Rats were randomly divided into four groups: Control group (n=15), low concentration (n=15), medium concentration (n=15), and high concentration (n=15). The low, medium, and high concentration groups were exposed to graphene oxide nanopowder 6 h/day for 5 days, while the control group received filtered fresh air. Animals were examined daily for evidence of exposure-related toxic reactions. Body weights were measured at the time of purchase, grouping, once during inhalation, and before postmortem.

Feed intake (g/rat/day) was measured once a week. After the 5 days of graphene oxide exposure, rats were allowed 1, 3,and 21 days (n = 5 per treatment group for each period) to examine clearance. All animal experiments were approved by the Institutional Animal Care and Use Committee of Hanyang University.

Organ Weights, Total Pathology and Histopathology

After blood collection, rats were euthanized with the anesthetic Entobar ® . followed by careful removal of the testes, heart, thymus, trachea, lungs, kidneys, spleen, liver, and brain. Organs were examined for gross lesions and then weighed and fixed in a 10% formalin solution containing neutral phosphate-buffered saline (PBS).

For histopathologic evaluation, the testes were fixed in a Bouin solution during killing, whereas the left lung was fixed in a 10% formalin solution (BBC Biochemical, Washington, DC) containing neutral phosphate-buffered saline under 25 cm water pressure. After fixation of the organs in 10% natural PBS for one week, they were then embedded in kerosene and stained with hematoxylin and eosin (BBC biochemical, Washington, DC). All animal organs were examined by light microscopy. The left lung was divided into three parts and examined.

Hematology and Blood Biochemistry

After an intraperitoneal injection of the anesthetic Entobar ® (1 ml/kg) and before euthanasia, blood samples were collected from the abdominal aorta into EDTA tubes for hematology assay and separation tubes for determination of blood biochemistry .

Blood was analyzed using a blood analyzer (Hitachi 7108, Hitachi, Tokyo, Japan), whereas hematology was analyzed using a blood cell counter (Hemavet 0950, CDC Tech., Dayton, OH). Blood coagulation was analyzed using blood coagulation equipment (ACL700, Instrumentation Laboratory, Bedford, MA).

Bronchoalveolar lavage (BAL) Cell Analysis and Measurement of Inflammatory Markers and Cytokines in BAL fluid

At sacrifice, the right lungs were injected four times with 3 ml aliquots of warm calcium- and magnesium-free phosphate-buffered saline (PBS) (pH 7.4). The BAL fluids were then centrifuged at 500 × g for 7 min, and the BAL cells were collected and resuspended in 1 ml of PBS for evaluation. Total cell counts were determined using a hemocytometer. Cells were first smears and then stained with Wright Giemsa Sure Stain to allow counts of total cells, macrophages, polymorphonuclear cells (PMNs), and lymphocytes.

Two hundred cells were evaluated for cell differentiation. In addition, BAL samples were also analyzed using a biochemical blood analyzer (Hitachi 7108, Hitachi, Japan) to determine the levels of lactate dehydrogenase (LDH), microalbumin (mALB), micro total protein (mTP), and blood urea nitrogen (BUN). Inflammatory cytokine (TNF-α, IL-1β) concentrations in BAL fluid were determined using a Quantikine Rat IL-1β/IL-1F2 immunoassay (R&D Systems, Inc., Minneapolis, MN) and a Quantikine Rat TNF-α immunoassay . measured (R&D Systems, Inc., Minneapolis, MN) according to the manufacturer’s instructions (principle: sandwich enzyme immunoassay).

Lung Deposition and Dose Calculation

Daily lung exposure per rat was estimated for 6 h continuous exposure, 1 min ventilation of 0.19 l/min ( Whalan et al., 2006 ) and the following aerosol properties (see Results section) : 203 nm particle MMAD, 2.01 GSD , 15.36% lung deposition efficiency based on MPPD (2002) (Multiple-Path Particle Dosimetry) Model v.2.0 and graphene oxide aerosol concentrations of 0.76, 2.60 and 9.78 mg/m 3 for the low, medium and high concentration, respectively.

The following Calculations were made:

  • Deposited daily dose (mg/day) = average graphene oxide aerosol concentration (mg/m 3 ) × minute volume (l/min = 0.06 m 3 /h) × exposure duration (h/day) × deposition efficiency (1).
  • Low dose deposition = 0.76 × (0.19 × 0.06) × 6 × 0.154 = 0.008 mg/day
  • Moderate dose deposition = 2.60 × (0.19 × 0.06) × 6 × 0.154 = 0.027 mg/day
  • High dose deposition = 9.78 × (0.19 × 0.06) × 6 × 0.154 = 0.103 mg/day
  • Cumulative dose (mg)/animal = deposited daily dose (mg/day) × number of days (5).
  • For low exposure: 0.008 × 5 = 0.04 mg
  • For medium exposure: 0.027 × 5 = 0.135 mg
  • At high exposure: 0.103 × 5 = 0.515 mg
Statistical analysis

Statistical analysis of the outcome parameters was performed using SPSS version 19 (SPSS Inc., Chicago, IL). Statistical analysis was performed by analysis of variance (ANOVA) after multiple comparison tests using Dunnett’s T3 method. The statistical significance level was set at p < 0.05, p < 0.01.

Results

Properties of graphene oxide nanopowder

The physicochemical properties of graphene oxide nanopowder are shown in Table 1 and Figure 1. The carbon and oxygen contents were 42-45% and 35-40%, respectively, based on thermogravimetric analysis (TGA), while the impurities were manganese <0.001% and sulfur <2.0%, based on inductively coupled plasma optical emission spectrometry ( ICP-OES). The thickness of graphene oxide was about 1 nm with 1-2 atomic layers and the lateral size was in the range of 0.5 to 5 μm.

Field emission TEM analysis of the graphene oxide nanopowder after aerosol generation revealed a stacked platelet structure with various thicknesses ranging from 5.94 to 209.1 nm (Figure 2). In addition, TEM-EDS analysis revealed the presence of two elements (i.e., C and O) (Figure 2). Table 2 shows the atomic percentages of the main graphene oxide components based on EDS analysis: carbon (72.69%), oxygen (27.31%).

Abb1
Fig. 1 Physicochemical properties of graphene oxide. XPS, X-ray photoelectron spectroscopy; XRD, X-ray diffraction; TGA, thermogravimetric analysis; FT-IR, Fourier transform infrared spectroscopy; UV-vis, ultraviolet-visible spectroscopy.
Abb2
Fig. 2 Analysis of graphene oxide by FE-TEM (AD) (field emission transmission electron microscopy) (×100,000). (EF) EDS spectrometer (energy dispersive spectroscopy).

Corona 1

Corona 3

 

Monitoring Chamber and Graphene Oxide Distribution

The target mass concentrations of graphene oxide nanopowder were 0.625 mg/m 3 , 2.5 mg/m 3 , and 10 mg/m 3 for the low, medium, and high concentrations, respectively. The mass concentrations delivered in the low, medium and high concentration chambers were 0.76 ± 0.10, 2.60 ± 0.19 and 9.78 ± 0.29 mg/m 3 .Table 3), respectively. The number concentrations in the chambers, measured with an optical particle counter (OPC), were 3.25 × 10 3 ± 1.18 × 10 2 , 6.30 × 10 3 ± 2.90 × 10 2 , and 9.97 × 10 3 ± 1.68 × 10 3 particles/ cm 3 for the low, medium, and high concentrations, respectively (Table 3). The particle number concentrations were maintained as shown in Figure 3.

The particle size distribution in the chambers was measured using a Scanning Mobility Particle Sizer (SMPS) (range 5.94 nm – 224.7 nm) and OPC (range 265 nm – 34 μm). The SMPS showed a peak at 22.5 nm, 25 nm, and 25.9 nm for the low, medium, and high concentrations, respectively (Figure 4A), while the OPC showed a peak at 265 nm, 365 nm, and 375 nm, respectively (Figure 4B). The mass median aerodynamic diameter (MMAD) measured with a 13-stage MOUDI 125NR cascade impactor was 203 nm with a GSD of 2.01 (Figure 5).

Figur 3
Figure 3: Particle number concentrations in graphene oxide aerosols in inhalation chambers during a 5-day exposure measured with the Scanning Mobility Particle Sizer (SMPS) (A) and the Optical Particle Counter (OPC) (B)
Figur 4
Figure 4: Particle size distribution of graphene oxide aerosols in inhalation chambers measured with SMPS (A) and OPC (B). The distributions were bimodal, with peak maxima at 20-30 nm (SMPS) and 300-400 nm (OPC), as shown in Table 3.
Abb 5
Figure 5: Mass median aerodynamic diameter (MMAD) (203 nm) and geometric standard deviation (GSD) (2.01) of aerosolized graphene oxide measured by MOUDI.

tisch 3

Elemental Carbon Analysis

The total EC was 0.18 ± 0.20 mg/cm 2 , 1.56 ± 0.54 mg/cm 2 , 3.34 ± 0.71 mg/cm 2 , and 7.25 ± 1.14 mg/cm 2 for the control, low, medium, and high concentration chambers, respectively (Table 4).

tabelle 4

Animal Observation, Feed Intake and Effect on Body and Organ Weight

No significant gross effects were observed during exposure. Also, no significant body weight losses or changes in food intake were observed during the exposure and recovery periods ( Table S2 and 3 ). However, right lung weights were significantly higher (P < 0.05) for the high concentration after 1 day, suggesting further verification of inflammation by histopathology and BAL cell and BAL fluid analyses ( Table S4 ).

Histopathology

While the number of alveolar macrophages with ingested graphene oxide increased in a concentration-dependent manner (Figure 6), a gradual clearance of graphene oxide was observed during the 21-day post-exposure period. Regardless, some macrophages with ingested graphene still persisted on day 21 of the postexposure period (Figure 6, red arrows).

No significant histopathologic observations were noted in peripheral airway epithelium, interstitial tissue, alveolar space, or vasculature in liver and kidneys. Light microscopic observation revealed no clear evidence of movement of graphene oxide ingested macrophages to lymph nodes adjacent to the bronchi. In addition, there was no significant histopathologic response of lung parenchyma, and even at low magnification, an exclusively adaptive response of lung macrophage clearance was noted (Figure 7). Histopathologic examination of the liver and kidney revealed no significant test article-induced lesions.

Abb 6
Figure 6: Lung histopathology at 1, 3, and 21 days. Panels are organized as day after exposure vs. concentration. Magnification 400x. None of the micrographs showed inflammation in bronchioles or perivascular regions. There were no fibrotic cell proliferations in interstitial tissues. Macrophages with ingested graphene oxide were detected in a concentration-dependent manner. No granulomatous appearance was observed at various concentrations throughout the post-exposure period. Red arrows indicate macrophages with ingested graphene oxide
Abb 7
Figure 7: Lung histopathology after 1, 3 and 21 days. Panels show microscopic images of control and high concentration at low magnification (100x). None of the microscopic images show inflammation in bronchioles or perivascular regions or lung parenchyma. Red arrows indicate macrophages with ingested graphene oxide.
Measurement of Inflammatory Markers and Cytokines

The differential cell count in the BAL showed no significant changes in the total number of cells, macrophages, lymphocytes, or PMN (Table 5). When the BAL inflammatory biomarkers were compared with the control group, a significant increase in mTP was observed on day 3 after exposure for the low concentration group.

Again, when compared with the control group, all exposed groups showed a consistent significant decrease in mALB at each time point after exposure, but no significant change in BUN or LDH. (Table 6). In addition, BAL fluid showed no significant changes in cytokines IL-1β or TNF-α during the post-exposure period (Table 6).

tabelle 5

tabelle 6

Effect on Blood Coagulation

Compared with the control group, none of the exposed groups showed any change in PT and APTT blood coagulation markers during the 21-day post-exposure period (Table 7).

tabelle 7

Hematology and Blood Biochemistry

Mean corpuscular hemoglobin concentration (MCHC) showed a significant decrease (P < 0.01) in the low concentration group after 1 day ( Table S7 ). In addition, MCHC showed a decrease (P < 0.05) in the medium- and high-concentration groups after 3 days but an increase (P < 0.05) in the high-concentration group after 21 days ( Table S8-9 ). . Mean platelet volume (MPV) showed an increase (P < 0.01) in the low concentration group after 1 day ( Table S7 ) .

The percentage of unstained cells (LUC) also increased (P < 0.01) in the low concentration group after 1 day and increased (P < 0.05) in the low and medium concentration groups after 3 days ( Table S7-8) . The absolute number of large unstained cells (abs luc) increased (P < 0.05) in the medium concentration group after 3 days ( Table S8 ). While LUC showed a consistent trend of significant increases across all time points after exposure, these changes were all within the normal range of numerical control values for this end point.

The medium concentration group showed lower cholesterol levels (CHO) compared to the low concentration group (p < 0.05) ( Table S10 ). Creatine level (CRE) also decreased (P < 0.05 – 0.01) in the medium and high concentration groups after 1 day ( Table S10 ). Inorganic phosphate (IP) decreased significantly (P < 0.05 – 0.01) in all exposed groups after 1 day and in the medium- and high-concentration groups (P < 0.05) after 3 days ( Table S10-11 ). Lactate dehydrogenase (LDH) level decreased (P < 0.05) in the high concentration group after 1 day and decreased (P < 0.01) in the medium and high concentration groups after 3 days ( Table S10-11). The high concentration group also showed lower LDH level than the low concentration group (p < 0.05) ( Table S10 ).

Magnesium level (MG) decreased (P < 0.01) in the medium and high concentration groups after 1 day and decreased (P < 0.01) in the low, medium and high concentration groups after 3 days . The medium and high concentration group also showed lower MG than the low concentration group (P < 0.01) ( Table S 10-12 ). Triglyceride (TG) level increased (p < 0.01) in the low concentration group after 1 day and increased (P < 0.01) in the high concentration group after 3 days ( Table S10-11) . While TG levels showed consistent significant increases across time points after exposure, these changes were within the normal range of numerical control values.

The level of glutamic oxidative transaminase (GOT) decreased (P < 0.01) in the low concentration group after 1 day and increased (P < 0.01) in the high concentration group after 3 days ( Table S11 ). The high concentration group also showed lower GOT compared with the low concentration group (p < 0.05) ( Table S10 ). Creatine kinase (CK) level decreased in the medium and high concentration groups after 3 days (P < 0.01) ( Table S11 ). The level of sodium (Na) decreased in the low concentration group after 3 days (P < 0.05) ( Table S11).

Albumin (ALB) and alkaline phosphatase (ALP) levels decreased in the medium and low concentration groups, respectively, after 21 days (P < 0.05) ( Table S12 ). Glucose level (GLU) increased in the medium and high concentration groups after 21 days (P < 0.01) ( Table S12 ). However, while consistent significant increases in IP, GOT, LDH, MG, CK were observed at all time points after exposure, these changes were all within the normal range of numerical control values ( Table S10-11 ). Consequently, no significant effects of toxicological significance on renal and hepatic hematological function were observed after graphene oxide exposure.

Discussion

The terms “safe innovation” and “safe by design” are currently widely used in the field of nanotechnology to advocate safety considerations early in the innovation process of nanomaterials and nanocapable products ( Park et al., 2017 ) . Graphene nanomaterials are indeed a good example of safe innovation or safe-by-design. Due to the strong interest in the commercial development of graphene-related nanomaterials and the increasing production trend, the assessment of the associated risks before the start of production is very important. A recent review of graphene-based nanomaterial toxicity studies in laboratory animals indicated potential behavioral, reproductive, and developmental toxicities and genotoxicities ( Ema et al., 2017 ).

While the acute inhalation toxicity of graphene oxide has been described as low ( Han et al, 2015 ), the repeated inhalation toxicity of graphene oxide has not yet been studied. Therefore, the current study, based on the short-term inhalation study by Ma-hock et al (2009), represents a first step in hazard assessment and range-finding for future subacute and subchronic studies. Consequently, based on hematological and biochemical analyses after graphene oxide exposure and during a post-exposure observation period, the current short-term inhalation study did not reveal significant systemic toxic effects of graphene oxide.

The results also showed a similar trend to a previous subacute graphene exposure study (Kim et al., 2016). Histopathological examination of the liver and kidney revealed no significant test article-relevant histopathological changes. In addition, no significant differences were observed in bronchoalveolar lavage cell differences, such as lymphocytes, macrophages, and PMNs. Evaluation of BAL fluid cytokines (ie IL-1β,TNF-α) also showed no significant concentration-dependent changes during the post-exposure period (Table 6).A spontaneous clearance response of graphene oxide ingested alveolar macrophages was observed in the lungs of all concentration groups throughout the 21-day post-exposure period.

Table 8 presents a comparison of toxicity results for graphene and graphene oxide in various short-term and subacute inhalation studies with graphene. The short-term graphene inhalation study by Shin et al, 2015 and the subacute graphene inhalation study by Kim et al, 2016 reported nearly identical toxicity results as the acute graphene oxide inhalation study by Han et al, 2015 and the current short-term graphene oxide inhalation study. However, another graphene nanoplate pharyngeal aspiration study with lateral sizes of < 2, 5, and 20 µm found increased inflammatory markers in BAL fluids when graphite nanoplates larger than 5 µm were used ( Roberts et al., 2016).

These different results could be due to the lateral size distribution of graphene and graphene oxide. When the lateral size was less than 5 µm, alveolar macrophages imaged with graphene and graphene oxide showed similar results with minimal toxicity. However, when the lateral size was larger than 5 μm, graphene induced an inflammatory response in BAL fluid after exposure ( Ma-hock et al., 2013 ). In the studies by Shin et al (2015) and Ma-hock et al (2013), animals were exposed to a maximum of 3 µg/m 3 and 10 µg/m 3 by inhalation to graphene nanoplates, respectively, and allowed to recover for 21 days. While the graphene nanoplate aerosols had a similar MMAD in each study, the inflammatory responses were determined by the different lateral sizes of the graphene nanoplates.

In addition, different delivery methods may also influence the inflammatory response. Intratracheal instillation (Shinwald et al., 2012) or pharyngeal aspiration techniques for delivery to the lungs of rats or mice may result in greater aggregation of nanomaterials and generally do not accurately reproduce the deposition patterns seen with inhalation exposure to dry aerosolized or nebulized suspensions of nanomaterials.

The main difference is that bolus exposure (instillation or aspiration) is a nonphysiological way to deliver a liquid-suspended material within fractions of a second at a very high dose rate.whereas physiological inhalation deposits aerosolized materials over a longer period of time (days, weeks, or months at low dose rates) (Oberdorster et al., 2015 ). Therefore, the inflammatory response induced after intratracheal instillation of graphene oxide and reduced graphene oxide in mice with an increased acute phase response along with serum amyloid A ( Bengston et al., 2017 ) was not observed in the current inhalation study, which did not show a marked inflammatory response.

tablle 8

Based on the authors’ short-term inhalation study, a subchronic inhalation study with graphene oxide nanoplates was also completed, and the results showed that macrophages had ingested particles at moderate and high concentrations as a spontaneous response to clearance. Therefore, the proposed NOAEL for the subchronic inhalation study was 3.02 mg/m 3 and no target organs were identified ( An et al., 2017 ).

Recently revised OECD test guidelines for subacute and subchronic inhalation toxicity testing now require lung exposure analysis to provide information on pulmonary deposition and particle retention in the lung at the end of exposure and at post-exposure observation intervals ( OECD 2017a ; 2017b ). In the current study, elemental carbon (EC) measurements of lung tissue from 1 day to 21 days were used to provide information on graphene oxide clearance. However, there was no available technology to analyze EC from lung tissue. This technology has since been developed by the current authors and will be used in our future inhalation studies of carbon nanomaterials.

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