An AOP-Based Integrated In Vitro and In Vivo Assessment of the Non-Genotoxic Carcinogenic Potential of Multi-Walled Carbon Nanotubes

Abstract

Multi-walled carbon nanotubes (MWCNTs) are increasingly incorporated into industrial and consumer products, raising concerns about potential carcinogenicity because their physicochemical properties vary widely among materials. Although Mitsui-7 has been classified as possibly carcinogenic to humans (IARC, Group 2B), the carcinogenic potential of domestically manufactured MWCNTs and the determinants underlying material-specific differences remain insufficiently characterized. Here, we applied an adverse outcome pathway (AOP)-oriented integrated testing strategy (ITS) to compare four domestically manufactured MWCNTs with Mitsui-7 using human bronchial epithelial BEAS-2B cells. Acute responses were assessed by measuring cytotoxicity and intracellular reactive oxygen species (ROS). Exposure concentrations for long-term studies were selected using range-finding assays, and cells were then exposed for four weeks at non-cytotoxic concentrations. Following chronic exposure, transformation-related phenotypes were evaluated using anchorage-independent growth, anchorage-dependent clonogenicity, wound healing migration, and Transwell–Matrigel invasion assays, and tumorigenic potential was examined in xenograft models using colony-derived cells. Highly aggregated MWCNTs elicited stronger oxidative stress and were associated with increased proliferation/clonal expansion, enhanced anchorage-independent colony formation, and increased tumor formation in vivo, whereas other materials showed more limited or endpoint-specific responses. Overall, the results indicate that MWCNT-associated carcinogenic potential is material-dependent rather than a uniform class effect and support the utility of an AOP-aligned ITS for nanosafety assessment and hazard differentiation of carbon-based nanomaterials.

1. Introduction

Multi-walled carbon nanotubes (MWCNTs) are increasingly incorporated into a wide range of industrial and consumer products, including smartphones, tablets, laptops, and wearable devices, owing to their exceptional mechanical strength, electrical conductivity, and durability. As the production and application of MWCNT-containing products continue to expand, concerns regarding potential adverse health effects associated with human exposure to MWCNTs have grown correspondingly.

MWCNTs are cylindrical carbon-based nanomaterials composed of multiple concentric graphene layers. Despite their advantageous physicochemical properties, accumulating evidence indicates that these same features may also contribute to adverse biological effects following inhalation or occupational exposure. Importantly, MWCNTs do not represent a uniform class of materials; rather, their physicochemical characteristics vary substantially depending on the synthesis method, such as chemical vapor deposition (CVD), arc discharge, or laser ablation [1,2]. These production-dependent differences directly influence critical parameters, including diameter, length, aspect ratio, rigidity, aggregation state, and structural integrity, all of which are known to modulate cellular interactions and biological responses [3,4].

Consistent with this physicochemical heterogeneity, MWCNTs have been reported to exhibit a broad spectrum of toxicological profiles, ranging from relatively low biological reactivity to pronounced pulmonary toxicity, genotoxicity, immunotoxicity, and carcinogenic potential [2,4,5,6,7,8,9]. Notably, certain MWCNTs, such as Mitsui-7, have been classified by the International Agency for Research on Cancer (IARC) as possibly carcinogenic to humans (Group 2B), underscoring the necessity for material-specific hazard evaluation rather than broad generalization across the entire MWCNT category. These observations highlight the critical importance of understanding how synthesis-dependent physicochemical properties contribute to differential biological and carcinogenic outcomes. Recent systematic reviews have synthesized extensive in vitro and in vivo evidence on CNT toxicology, underscoring variability in biological responses depending on dose, dispersion state, cell type, and material characteristics [10,11].

Despite growing concerns regarding MWCNT-associated health risks, relatively limited information is available regarding how variations in physicochemical properties influence carcinogenic potential, particularly for domestically manufactured MWCNTs. Therefore, the objective of the present study was to systematically evaluate and compare the carcinogenic potential of four domestically manufactured MWCNTs with that of Mitsui-7, a well-characterized reference material associated with carcinogenic risk. By focusing on long-term exposure-induced malignant transformation phenotypes, this study aimed to determine whether domestically produced MWCNTs exhibit carcinogenic properties comparable to, or distinct from, those of Mitsui-7.

To investigate malignant transformation induced by nanomaterial exposure, the experimental strategy was designed based on the multistage model of carcinogenesis, which comprises initiation, promotion, and progression [12,13]. The initiation stage involves genotoxic events leading to irreversible DNA damage, whereas the promotion and progression stages are largely driven by non-genotoxic mechanisms that promote sustained cell proliferation, phenotypic alteration, and malignant conversion [14]. Although numerous studies have reported genotoxic effects of MWCNTs [7,15], comparatively, few investigations have addressed non-genotoxic carcinogenic processes associated with chronic nanomaterial exposure. Consequently, toxicological evaluation strategies capable of interrogating the promotion and progression stages of carcinogenesis are critically needed.

In this study, human bronchial epithelial BEAS-2B cells were employed as a model system. BEAS-2B cells are non-tumorigenic, immortalized human airway epithelial cells that have been extensively used to investigate carcinogenic transformation and tumorigenic potential in vitro [9,16,17,18,19]. To determine whether chronic exposure to MWCNTs induces malignant phenotypic transformation in normal epithelial cells, a comprehensive testing strategy was applied based on previously established approaches and aligned with an adverse outcome pathway (AOP) framework [20].

Within this AOP-oriented integrated testing strategy (ITS), early cellular key events were first assessed by evaluating acute cytotoxicity and oxidative stress following MWCNT exposure. Subsequently, BEAS-2B cells were chronically exposed to MWCNTs for four weeks at non-cytotoxic concentrations determined using colony formation efficiency (CFE) and crystal violet (CV) assays. To capture key events associated with the promotion and progression stages of carcinogenesis, malignant transformation-related phenotypes were then evaluated, including anchorage-independent colony formation, anchorage-dependent clonogenic growth, cell migration, and invasive capacity. Finally, to confirm whether the in vitro transformation phenotypes translated into adverse outcomes at the organism level, colony-derived BEAS-2B cells were harvested from soft agar and subjected to xenograft tumorigenicity assays in nude mice.

By integrating these cellular and in vivo endpoints along the AOP continuum, the present study systematically assessed and compared the carcinogenic potential of four domestically manufactured MWCNTs relative to Mitsui-7. This integrated approach enables a final determination of whether domestic MWCNTs exhibit carcinogenic properties comparable to, or distinct from, those of a reference MWCNT classified as possibly carcinogenic to humans. Figure 1 illustrates the AOP-based integrated testing strategy used in this study, showing the progression from early cellular responses to malignant transformation and in vivo tumorigenicity following chronic MWCNT exposure.

2. Materials and Methods

2.1. Nanomaterial Preparation and Dispersion

Except for Mitsui-7 (Tokyo, Japan), which was manufactured in Japan, four MWCNTs were manufactured in the Republic of Korea and supplied by six different manufacturers. Selected carbon nanotubes were obtained from the same supplier as those used in the study by Lee et al. [3] and corresponds to previously characterized reference materials. Detailed physicochemical characteristics of each nanomaterial are provided in

Table A1. Physicochemical properties of MWCNT samples [3].

Samples	Diameter (nm)	Length (μm) a	Raman (IG/ID)	BET (m2/g)	Purity (%) b	Endotoxin (EU/mL)
MWCNT 1	15.64 ± 0.1	10–50	1.05	194.03	95	<0.1
MWCNT 2	7.75 ± 0.1	1–25	0.64	675.44	99.1–99.4	<0.1
MWCNT 3	16.37 ± 0.4	-	0.85	218.26	>90	<0.1
MWCNT 4	16.7 ± 0.2	−200	0.92	224.90	95	<0.1
Mitsui-7	58.3 ± 1.0	1–19	1.01	28.2	>95	<0.1

^a the length of highly entangled fibers could not be accurately measured because of their pronounced curliness. ^b purity and fiber length values were based on specifications supplied by the manufacturer.

.

The nanomaterials were dispersed in 10 mL of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS; HyClone™, Cytiva, Marlborough, MA, USA) using Pluronic^® F-127 as a dispersant at a final concentration of 0.05% (w/v). Stock suspensions were prepared at a particle concentration of 100 µg/mL. Dispersion was performed by probe sonication using a QSonica Q700 sonicator (Qsonica LLC, Newtown, CT, USA) with a 6.4 mm probe, operated at 700 W and 80 kHz for a total of 10 min with a pulse mode of 10 s and 10 s off. Immediately prior to each experiment, the stock suspensions were freshly diluted in complete DMEM to the designed working concentrations. Due to the high aspect ratio and fibrous morphology of MWCNTs, conventional particle size characterization techniques such as dynamic light scattering (DLS) and zeta potential analysis were not applicable in this study. These techniques are based on assumptions of spherical particle geometry and are known to yield unreliable or misleading results for elongated, fiber-like nanomaterials with high aspect ratios. The stability of the dispersed MWCNT suspensions was evaluated under actual in vitro exposure conditions. Specifically, immediately after dispersion and over a 24 h period corresponding to the exposure duration used in the cell-based assays, the suspensions were visually inspected and microscopically examined to assess aggregation and sedimentation behavior. Dispersion stability was qualitatively monitored immediately after sonication and up to 72 h under cell culture conditions by visual inspection and light microscopy. While gradual sedimentation was observed over time, dispersion was maintained during the initial 24 h exposure period, corresponding to acute cytotoxicity and ROS assays.

Accordingly, alternative image-based characterization approaches, including FE-SEM-derived morphological assessment, were employed to qualitatively evaluate fiber morphology, aggregation state, and dispersion behavior.

2.2. Characterization of Multi-Walled Carbon Nanotubes (MWCNTs)

Dispersed MWCNTs were diluted to the highest concentrations used in the experiments. Aliquots (10 µL) of each suspension were deposited onto the center of an Isopore™ membrane filter (Millipore, Darmstadt, Germany) and allowed to air-dry at room temperature. After drying, the membrane filters were mounted on carbon adhesive tape and sputter-coated with platinum to enhance surface conductivity. The morphological characteristics of the nanomaterials were examined using field emission scanning electron microscopy (FE-SEM; S8000, TESCAN, Brno, Czech Republic). Elemental composition was analyzed by energy-dispersive X-ray spectroscopy (EDS) integrated into the FE-SEM system.

2.3. Cell Culture and Exposure

The human bronchial epithelial cell line BEAS-2B (CRL-9609; American Type Culture Collection, ATCC, Manassas, VA, USA), an SV40 large T antigen–immortalized human bronchial epithelial cell line, was obtained from Coram Biotech (Seoul, Republic of Korea). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and maintained at 37 °C in a humidified atmosphere containing 5% CO₂. As BEAS-2B is a commercially available, established immortalized cell line, its use does not require approval from an institutional review board or ethics committee. For the assessment of acute cytotoxicity and oxidative stress, MWCNTs and CNFs were dispersed in DMEM containing 10% FBS and applied to BEAS-2B cells at concentrations of 1, 5, 10, and 20 µg/mL for 24 h. For dose-selection experiments, MWCNTs were diluted in 10% DMEM and administered at concentrations of 1, 5, 10, and 20 for 24 h. For dose selection experiments, MWCNTs were diluted in 10% FBS-DMEM and administered at concentrations of 1, 5, and 10 µg/mL for 3 days. Based on these results, chronic exposure experiments were conducted by continuously exposing BEAS-2B cells to MWCNTs at concentrations of 1 and 10 µg/mL in 10% FBS-DMEM for 4 weeks, corresponding to approximately 10 serial passages. During chronic exposure, the culture medium containing nanomaterials was replaced at each passage.

2.4. WST-1 Cytotoxicity Assay

Cytotoxicity was evaluated using a WST-1 assay (CellVia™ Enhanced Cell Viability Assay Kit, Ab FRONTIER, Seoul, Republic of Korea). BEAS-2B cells were seeded at a density of 5 × 10⁴ cells per well in 96-well plates containing 200 µL of DMEM supplemented with 10% FBS and incubated for 24 h at 37 °C in a humidified atmosphere with 5% CO₂. Cells were then exposed to five MWCNT samples, diluted in 10% FBS-DMEM, for 24 h. Following exposure, the cells were washed twice with Phosphate-buffered saline (PBS), and fresh medium containing WST-1 reagent was added at a ratio of 100 µL medium to 10 µL WST-1. After incubation for 30–120 min at 37 °C, absorbance was measured at 540 nm using a microplate reader (TECAN, Männedorf, Switzerland). Cell viability was expressed as a percentage relative to untreated control cells. All experiments were performed in triplicate and repeated at least three times independently.

2.5. Measurement of Reactive Oxygen Species (ROS)

Intracellular ROS generation was assessed using the dichlorofluorescin diacetate (DCFDA) assay (Thermo Fisher, Waltham, MA, USA). BEAS-2B cells were seeded at a density of 2 × 10⁵ cells per well in 6-well plates containing 1.5 mL of DMEM supplemented with 10% FBS and incubated for 24 h at 37 °C in a humidified atmosphere with 5% CO₂. Cells were then exposed to MWCNTs diluted in 10% FBS-DMEM for 24 h. Following exposure, cells were washed twice with PBS, detached using trypsin-EDTA, and resuspended in 10% FBS-DMEM. The cell suspensions were incubated with 20 µM DCFDA in PBS for 30 min at 37 °C in the dark. After incubation, cells were centrifuged and washed twice with PBS. Fluorescence intensity was measured at excitation and emission wavelengths of 485 and 528 nm, respectively, using a multimode microplate reader (TriStar² LB 942, Berthold Technologies, Bad Wildbad, Germany). All measurements were performed in triplicate.

2.6. Range Finding Assay by CV and CFE

BEAS-2B cells were cultured to approximately 80% confluence, detached using trypsin, and resuspended in DMEM supplemented with 5% fetal bovine serum (FBS). Cells were then seeded either into 6-well plates at a density of 3 × 10³ cells per well in 1.5 mL of 5% FBS-DMEM for the crystal violet (CV) assay, or into 60 mm culture dishes at a density of 200 cells per dish in 4 mL of 5% FBS-DMEM for the colony formation efficiency (CFE) assay. After 24 h of incubation to allow cell attachment, the culture medium was replaced with fresh 5% FBS-DMEM containing MWCNTs at concentrations ranging from 1 to 10 µg/mL. Cells were exposed to MWCNTs for 72 h, after which they were washed twice with phosphate-buffered saline (PBS) and further cultured in fresh DMEM supplemented with 10% FBS. Following an additional incubation period of 3 days for the CV assay or 5 days for the CFE assay, cells were fixed and stained with 0.1% crystal violet or 0.04% Giemsa solution, respectively. For the CV assay, the bound dye was solubilized using 1.5 mL of extraction solution composed of ethanol, distilled water, and 1 M HCl (50:49:1, v/v/v), and absorbance was measured at 540 nm. For the CFE assay, stained colonies consisting of ≥50 cells or with diameters ≥ 2 mm were counted using an image-based stereological analysis system equipped with an upright microscope (Olympus CX31, Japan) and a CCD digital camera (IMT scan cooled model, IMT i-Solution Inc., Jena, Germany).

2.7. Anchorage-Independent Colony Formation Assay

Anchorage-independent growth was evaluated using a soft agar colony formation assay. BEAS-2B cells were cultured in 100 mm dishes and chronically exposed to MWCNTs for 4 weeks prior to the assay. For the base layer, 1% agar prepared in DMEM supplemented with 10% FBS was dispensed into 12-well plates and allowed to solidify at room temperature. For the top layer, exposed cells were suspended at a density of 3 × 10³ cells per well in 0.7% agar prepared in 10% FBS-DMEM and overlaid onto the solidified base agar. After solidification of the top agar, 10% FBS-DMEM was added to each well. Cultures were maintained for 3 weeks at 37 °C in a humidified atmosphere with 5% CO₂, with medium replacement every 3 days. At the end of the incubation period, colonies were stained with nitro blue tetrazolium (0.5 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) for visualization. Colonies with diameters ≥ 2 μm or 50 μm were counted under a stereomicroscope. Colony numbers were expressed as a percentage relative to untreated control cells. All experiments were performed in triplicate.

2.8. Wound Healing Migration Assay

Cell migratory capacity was evaluated using a wound healing assay. BEAS-2B cells were cultured in 100 mm culture dishes and chronically exposed to MWCNTs for 4 weeks. Following exposure, cells were harvested, resuspended in serum-free DMEM, and seeded into 12-well plates at a density of 2 × 10⁵ cells in 300 μL/well. After incubation for 24 h to allow cell attachment, a straight scratch was generated across the center of each well using a sterile pipette tip. Detached cells were gently removed by washing with PBS, and fresh serum-free DMEM was added. Wound closure was monitored at 0 and 48 h after scratching using an inverted microscope (CKX53, Olympus, Tokyo, Japan). The migration distance was quantified using the i-Solution Auto Plus ×64 software (version 26.1, IMT i-Solution Inc., Burnaby, BC, Canada). Migration was quantified as the percentage of wound closure relative to the initial wound area. All experiments were performed in triplicate.

2.9. Transwell–Matrigel Invasion Assay

Cell invasive potential was evaluated using a Transwell chamber assay. The upper surfaces of Transwell inserts were coated with Matrigel (Corning, New York, NY, USA) diluted 1:10 in culture medium and allowed to polymerize at 37 °C. BEAS-2B cells previously exposed to MWCNTs for 4 weeks were harvested, resuspended in serum-free DMEM, and seeded into the upper chamber at a density of 5 × 10⁵ cells in 300 μL per well. The lower chamber was filled with DMEM supplemented with 10% FBS to serve as a chemoattractant. After incubation for 48 h at 37 °C in a humidified 5% CO₂ atmosphere, non-invading cells on the upper surface of the membrane were gently removed using a cotton swab. Cells that had invaded the lower surface were fixed and stained with crystal violet. The stained cells were solubilized using an extraction buffer composed of ethanol, distilled water, and 1 M HCl (50:49:1, v/v/v). Absorbance was measured at 540 nm using a microplate reader to quantify cell invasion. Invasion was expressed as a percentage relative to untreated control cells. All experiments were performed in triplicate.

2.10. Proliferation and Colony Formation of Colony-Derived Cells Under Anchorage-Dependent Conditions

BEAS-2B cells chronically exposed to MWCNTs for 4 weeks were subjected to an anchorage-independent colony formation. Colonies were harvested, and both parental BEAS-2B cells and colony-derived cells were seeded into 96-well plates at 0.5 × 104 cells per well in 200 µL of complete medium. Cell proliferation was assessed at 24, 48, 72, and 96 h using a WST-1 assay, as described in Section 2.4 (WST-1 cytotoxicity assay).

To evaluate anchorage-dependent colony-forming ability, anchorage-independent colonies generated after 4 weeks of chronic exposure were harvested. Colony-derived cells and parental BEAS-2B cells were seeded in 6-well plates at a density of 300 cells per 4 mL of 10% FBS-DMEM. After 7 days of culture, colonies with a diameter ≥ 50 µm were counted under a stereomicroscope. Proliferation and colony formation were expressed relative to parental BEAS-2B control cells. All experiments were performed in triplicate.

2.11. Xenograft Tumorigenicity Assay

All animal procedures were conducted in an AAALAC-accredited facility (Biotoxtech Co., Ltd., Cheongju, Republic of Korea) in accordance with the Guide for the Care and Use of Laboratory Animals. The experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Biotoxtech (Approval No. 200454, “Evaluation of the Carcinogenic Potential of MWCNTs Using a Xenograft Model”). Six-week-old male athymic nude mice were purchased from Koatech (Gyeonggi-do, Korea) and acclimated for 7 days prior to the experiment. Animals were housed under controlled environmental conditions (temperature, 23 ± 3 °C; relative humidity, 55 ± 5%; 12 h light/dark cycle) with ad libitum access to γ-ray-sterilized rodent chow (2.0 Mrad, EP pellets) and autoclaved tap water. Transformed BEAS-2B cells were harvested and resuspended in sterile PBS. A total of 2 × 10⁶ cells in 100 µL were subcutaneously injected into the flanks of each mouse. Tumor growth was monitored at regular intervals using an external caliper, and tumor volume was calculated using the following formula:

$$Tumorvolume\left({mm}^3\right)={\displaystyle \frac{1}{2}}\times \left(length\times widt{h}^2\right).$$

On day 16 after cell injection, mice were euthanized, and tumors were excised and weighed.

2.12. Statistical Analysis

All experiments were conducted with at least three independent replicates. Data are presented as mean ± standard deviation (SD), calculated using SigmaPlot 12.0 (Systat Software Inc., San Jose, CA, USA) and Microsoft Excel 2019 (Microsoft Corporation, Redmond, WA, USA). Differences among experimental groups were evaluated using one-way analysis of variance (ANOVA). When appropriate, Dunnett’s multiple comparison test was applied to compare treatment groups with the corresponding control. Homogeneity of variance was assessed using Levene’s test. If the assumption of equal variances was violated, Welch’s ANOVA was used, followed by the Games-Howell post hoc test. A probability value of p < 0.05 was considered to indicate statistical significance. Levels of significance are denoted as * p < 0.05 and ** p < 0.01. All statistical tests were two-tailed. Statistical analyses for in vitro and in vivo experiments were performed separately.

3. Results

3.1. Characterization of MWCNTs

The morphological characteristics of multi-walled carbon nanotubes (MWCNTs) were examined using field emission scanning electron microscopy (FE-SEM; S8000, TESCAN, Brno, Czech Republic) to evaluate their aggregation behavior and fiber morphology. As shown in Figure 2, all MWCNT samples exhibited varying degrees of aggregation and distinct morphological features.

Among the tested materials, MWCNT 3 (Figure 2c) exhibited the most pronounced aggregation, forming densely packed and highly entangled network structures. MWCNT 2 and MWCNT 4 (Figure 2b,d) showed comparable aggregation patterns, characterized by partially bundled and moderately entangled fiber structures. In contrast, MWCNT 1 (Figure 2a) appeared relatively less aggregated, with more discernible individual fibers, although the nanotubes remained highly curved and entangled. Mitsui-7 displayed a distinct morphological profile compared with the other MWCNT samples, characterized by reduced aggregation and a straighter, more elongated fiber morphology with lower curvature and limited entanglement. These observations indicate marked differences in aggregation density and fiber straightness among the tested MWCNTs.

Elemental composition analysis was performed using energy-dispersive X-ray spectroscopy (EDS), confirming that all MWCNT samples were predominantly composed of carbon (Figure S1). Minor peaks corresponding to sodium (Na) and Chlorine (Cl) were detected and are attributable to residual components of the cell culture medium used during dispersion. Nitrogen (N) signals were also observed, which are presumed to originate from FBS components. Overall, the EDS results verify the carbon-based nature of the tested MWCNTs and indicate the presence of medium-related residues following sample preparation. Importantly, all MWCNT samples were dispersed using identical dispersion protocols and exposure conditions, ensuring that differences in aggregation behavior were not attributable to experimental handling. FE-SEM analysis of the pristine materials revealed distinct and reproducible aggregation and fiber entanglement patterns among the MWCNTs prior to biological testing, indicating that these features represent intrinsic material properties.

Figure 2. Field Scanning electron microscopy (SEM) images of the tested multi-walled carbon nanotubes (MWCNTs). Representative SEM images of (a) MWCNT 1, (b) MWCNT 2, (c) MWCNT 3, (d) MWCNT 4, and (e) Mitsui-7 are shown at low and high magnification. All samples exhibited aggregation to varying extents; however, distinct differences in aggregation density and fiber morphology were observed. MWCNT 3 showed the most pronounced aggregation, forming densely packed and highly entangled network structures. MWCNT 2 and MWCNT 4 exhibited moderate aggregation with partially bundled and entangled fibers, whereas MWCNT 1 appeared relatively less aggregated with more discernible individual fibers, despite maintaining a highly curved and entangled morphology. In contrast, Mitsui-7 displayed reduced aggregation and a straighter, more elongated fiber morphology with lower curvature and limited entanglement compared with the other MWCNT samples. The left and right panels correspond to low-magnification (×2.5 k–10 k) and high-magnification (×30 k–100 k) images, respectively.

Figure 2. Field Scanning electron microscopy (SEM) images of the tested multi-walled carbon nanotubes (MWCNTs). Representative SEM images of (a) MWCNT 1, (b) MWCNT 2, (c) MWCNT 3, (d) MWCNT 4, and (e) Mitsui-7 are shown at low and high magnification. All samples exhibited aggregation to varying extents; however, distinct differences in aggregation density and fiber morphology were observed. MWCNT 3 showed the most pronounced aggregation, forming densely packed and highly entangled network structures. MWCNT 2 and MWCNT 4 exhibited moderate aggregation with partially bundled and entangled fibers, whereas MWCNT 1 appeared relatively less aggregated with more discernible individual fibers, despite maintaining a highly curved and entangled morphology. In contrast, Mitsui-7 displayed reduced aggregation and a straighter, more elongated fiber morphology with lower curvature and limited entanglement compared with the other MWCNT samples. The left and right panels correspond to low-magnification (×2.5 k–10 k) and high-magnification (×30 k–100 k) images, respectively.

3.2. WST-1 Cytotoxicity and Reactive Oxygen Species (ROS) Generation in BEAS-2B Cells Exposed to MWCNTs

The cytotoxic effects of MWCNTs on BEAS-2B cells were evaluated after 24 h exposure at concentrations ranging from 1 to 20 μg/mL using the WST-1 assay (Figure 3a). All tested MWCNT samples exhibited a concentration-dependent decrease in cell viability compared with untreated control cells. At the highest exposure concentration (20 μg/mL), cell viability ranged from approximately 44% to 70% for MWCNTs 1–4, whereas Mitsui-7 exhibited a cell viability of approximately 64%. Statistically significant cytotoxic effects were generally observed at concentrations of 5–10 μg/mL and above for most MWCNT samples, indicating differences in cytotoxic potency among the tested materials. These findings are consistent with previous reports by Patlolla et al. [15], who demonstrated a marked decrease in cell viability following MWCNT exposure in a genotoxicity study.

Oxidative stress, characterized by excessive intracellular ROS production, is known to induce cellular damage to lipids, proteins, and DNA [21]. In the present study, intracellular ROS generation in BEAS-2B cells following 24 h exposure to MWCNTs was quantified using the DCFDA assay (Figure 3b). Exposure to MWCNTs resulted in a concentration-dependent increase in ROS levels. At 20 µg/mL, MWCNT 3 and MWCNT 4 markedly elevated ROS levels to approximately 250% and 220% of the control, respectively (** p < 0.01), whereas MWCNT 1 and MWCNT 2 induced moderate increases of approximately 180–200% relative to the control (* p < 0.05 and ** p < 0.01). In contrast, Mitsui-7 induced a comparatively modest increase in ROS generation, reaching approximately 140–160% of control levels even at the highest concentration tested (* p < 0.05), which was significantly lower than that observed for MWCNT 3 and MWCNT 4. These results indicate that the magnitude of oxidative stress induction varies substantially among MWCNTs and is strongly material-dependent rather than a uniform class effect.

Collectively, these results demonstrate that MWCNT exposure significantly induces oxidative stress in BEAS-2B cells. Notably, MWCNTs that produced stronger cytotoxic effects also tended to elicit higher levels of ROS generation, suggesting a close association between oxidative stress and MWCNT-induced cytotoxicity.

Figure 3. Effects of multi-walled carbon nanotubes (MWCNTs) on cell viability and intracellular reactive oxygen species (ROS) generation in BEAS-2B cells. Cell viability (a) and intracellular reactive oxygen species (ROS) generation (b) were assessed using the WST-1 and DCFDA assays, respectively, following exposure to MWCNTs at concentrations ranging from 1 to 20 µg/mL. BEAS-2B cells were cultured in DMEM supplemented with 10% FBS and incubated with the indicated concentrations of MWCNTs at 37 °C for 24 h. Cell viability and ROS levels are expressed as percentages relative to untreated control cells. For both assays, data represent the mean ± standard deviation (SD) from three independent experiments. Statistical significance compared with the control group is indicated as * p < 0.05 and ** p < 0.01.

Figure 3. Effects of multi-walled carbon nanotubes (MWCNTs) on cell viability and intracellular reactive oxygen species (ROS) generation in BEAS-2B cells. Cell viability (a) and intracellular reactive oxygen species (ROS) generation (b) were assessed using the WST-1 and DCFDA assays, respectively, following exposure to MWCNTs at concentrations ranging from 1 to 20 µg/mL. BEAS-2B cells were cultured in DMEM supplemented with 10% FBS and incubated with the indicated concentrations of MWCNTs at 37 °C for 24 h. Cell viability and ROS levels are expressed as percentages relative to untreated control cells. For both assays, data represent the mean ± standard deviation (SD) from three independent experiments. Statistical significance compared with the control group is indicated as * p < 0.05 and ** p < 0.01.

3.3. Selection of Exposure Concentrations for Long-Term MWCNTs Exposure Based on CV and CFE Assays

To determine appropriate exposure concentrations for subsequent long-term experiments, range-finding assays were performed using crystal violet (CV) and colony formation efficiency (CFE) assays. As shown in Figure 3, BEAS-2B cells exposed to MWCNTs exhibited material-dependent cytotoxic responses as assessed by the WST-1 assay. In comparison, the CV and CFE assays revealed both overlapping and distinct response patterns relative to those observed in the WST-1 cytotoxicity assay (Figure 4).

No statistically significant reduction in cell viability was observed for MWCNT 1 and MWCNT 3, whereas MWCNT 2, MWCNT 4, and Mitsui-7 induced a dose-dependent decrease in cell viability. Based on these findings, the no observable effect level (NOEL) and lowest observable effect level (LOEL) were determined for each MWCNT (Table 1).

For MWCNT 1, neither cytotoxicity nor inhibition of colony formation was observed up to 10 µg/mL, and thus both the NOEL and LOEL were considered to be above this concentration. For MWCNT 2, the NOEL and LOEL were identified as 1 µg/mL and 5 µg/mL, respectively, in both the CV and CFE assays. In the case of MWCNT 3, the CV assay indicated a NOEL of 1 µg/mL and a LOEL of 5 µg/mL, whereas the CFE assay suggested a NOEL below 1 µg/mL and a LOEL of 1 µg/mL.

For MWCNT 4, the CV assay showed a NOEL of 5 µg/mL and a LOEL of 10 µg/mL. In contrast, the CFE assay revealed no significant inhibition of colony formation by MWCNT 4, indicating that both the NOEL and LOEL were above 10 µg/mL. For Mitsui-7, no detectable cytotoxic effects were observed in the CV assay up to 10 µg/mL; however, the CFE assay demonstrated a significant, concentration-dependent suppression of colony formation, with the NOEL and LOEL determined to be 1 µg/mL and 5 µg/mL, respectively.

Based on the combined CV and CFE results and applying the criterion that cell death remained below 20%, exposure concentrations of 1 µg/mL and 10 µg/mL were selected as the low- and high-dose conditions, respectively, for subsequent long-term MWCNT exposure experiments. Although marked reductions in cell viability or colony formation to below 50% of control levels were observed for MWCNT 2 and for Mitsui-7 in the CFE, a unified exposure concentration range of 1 and 10 µg/mL was adopted to enable consistent comparison across all five MWCNT samples.

Accordingly, BEAS-2B cells were chronically exposed to MWCNTs for 4 weeks using these selected concentrations to evaluate long-term exposure-induced malignant phenotypes.

Figure 4. Range-finding assays for MWCNT exposure in BEAS-2B cells using crystal violet (CV) and colony formation efficiency (CFE) assays. BEAS-2B cells were seeded in 6-well plates for the crystal violet (CV) assay or in 60 mm culture dishes for the colony formation efficiency (CFE) assay and exposed to MWCNTs at the indicated concentrations for 72 h. Following exposure, the medium was replaced with fresh medium, and cells were further incubated for 3 days (CV) or 5 days (CFE). Cells were subsequently stained with crystal violet (CV) or Giemsa stain (CFE). Cytotoxic effects were expressed as percentages relative to untreated control cells. Data represents the mean ± standard deviation (SD) of three independent experiments. Statistical significance compared with the control group is indicated as * p < 0.05 and ** p < 0.01.

Figure 4. Range-finding assays for MWCNT exposure in BEAS-2B cells using crystal violet (CV) and colony formation efficiency (CFE) assays. BEAS-2B cells were seeded in 6-well plates for the crystal violet (CV) assay or in 60 mm culture dishes for the colony formation efficiency (CFE) assay and exposed to MWCNTs at the indicated concentrations for 72 h. Following exposure, the medium was replaced with fresh medium, and cells were further incubated for 3 days (CV) or 5 days (CFE). Cells were subsequently stained with crystal violet (CV) or Giemsa stain (CFE). Cytotoxic effects were expressed as percentages relative to untreated control cells. Data represents the mean ± standard deviation (SD) of three independent experiments. Statistical significance compared with the control group is indicated as * p < 0.05 and ** p < 0.01.

Table 1. No-observable-effect level (NOEL) and lowest-observable-effect level (LOEL) values for each MWCNT, determined by crystal violet (CV) and colony formation efficiency (CFE) assays.

Test Chemicals (µg/mL)		MWCNT1	MWCNT2	MWCNT3	MWCNT4	Mitsui 7
CV	NOEL	10<	1	1	5	10<
	LOEL	10<	5	5	10	10<
CFE	NOEL	10<	1	1	10<	1
	LOEL	10<	5	1	10<	5

Table 1. No-observable-effect level (NOEL) and lowest-observable-effect level (LOEL) values for each MWCNT, determined by crystal violet (CV) and colony formation efficiency (CFE) assays.

Test Chemicals (µg/mL)		MWCNT1	MWCNT2	MWCNT3	MWCNT4	Mitsui 7
CV	NOEL	10<	1	1	5	10<
	LOEL	10<	5	5	10	10<
CFE	NOEL	10<	1	1	10<	1
	LOEL	10<	5	1	10<	5

3.4. Anchorage-Independent Colony Formation as an Indicator of Malignant Transformation

Anchorage-independent colony formation is considered a stringent indicator of malignant transformation, as it reflects the ability of cells to proliferate without attachment to a solid substrate, a key hallmark of oncogenic transformation [22]. In this study, anchorage-independent growth was assessed using a soft agar colony formation assay to evaluate malignant phenotypic changes induced by long-term MWCNTs exposure.

BEAS-2B cells chronically exposed to MWCNTs for 4 weeks exhibited a significant, dose-dependent increase in anchorage-independent colony formation compared with untreated control cells (Figure 5a,b). Quantitative analysis demonstrated that at the lower exposure concentration (1 μg/mL), significant induction of anchorage-independent colony formation was observed only for MWCNT1 and MWCNT 4, corresponding to approximately 3- and 4-fold increases over control levels, respectively. In contrast, MWCNT 2 and MWCNT 3 at 10 μg/mL induced the most pronounced responses, resulting in approximately 23-fold and 50-fold increases in colony formation, respectively.

The reference material Mitsui-7 exhibited a significant increase in colony formation only at the higher concentration (10 μg/mL), while no significant effect was detected at 1 μg/mL. Collectively, these findings indicate that chronic exposure to specific MWCNT variants, particularly MWCNT2 and MWCNT3, markedly enhances anchorage-independent growth, suggesting an increased malignant transformation potential in non-tumorigenic human bronchial epithelial cells. Notably, several domestically manufactured MWCNTs induced anchorage-independent growth comparable to or exceeding that of Mitsui-7, underscoring pronounced material-specific differences in carcinogenic potential rather than a uniform class effect.

Figure 5. Anchorage-independent agar colony formation in BEAS-2B cells following chronic MWCNTs exposure. BEAS-2B cells were exposed to MWCNTs for 4 weeks and assessed using a soft agar assay. (a) Representative images of anchorage-independent colonies formed after exposure to 1 and 10 μg/mL MWCNTs. (b) Quantification of colony formation expressed as fold change relative to untreated control. Colonies ≥ 50 μm were counted. Data are presented as mean ± SD (n = 3). Statistical significance compared with the control group is indicated as * p < 0.05 and ** p < 0.01.

Figure 5. Anchorage-independent agar colony formation in BEAS-2B cells following chronic MWCNTs exposure. BEAS-2B cells were exposed to MWCNTs for 4 weeks and assessed using a soft agar assay. (a) Representative images of anchorage-independent colonies formed after exposure to 1 and 10 μg/mL MWCNTs. (b) Quantification of colony formation expressed as fold change relative to untreated control. Colonies ≥ 50 μm were counted. Data are presented as mean ± SD (n = 3). Statistical significance compared with the control group is indicated as * p < 0.05 and ** p < 0.01.

3.5. Migration and Invasion of BEAS-2B Cells Following Chronic Exposure to MWCNTs

3.5.1. Cell Migration Assessed by the Wound Healing Assay

Cell migration, defined as the directed movement of cells from one location to another, is a key phenotypic feature associated with malignant progression [23]. The migratory capacity of BEAS-2B cells following chronic exposure to MWCNTs was evaluated using a wound healing assay.

As shown in Figure 6, control cells exhibited limited wound closure over 48 h, whereas cells chronically exposed to specific MWCNTs displayed enhanced migratory activity in a material- and concentration-dependent manner. Quantitative analysis revealed that MWCNT 1 induced a significant increase in wound closure at 1 μg/mL, while MWCNT 2 showed pronounced and statistically significant increases at 10 μg/mL.

In contrast, MWCNT 3 and MWCNT 4 did not induce enhanced migration at either concentration, despite their strong effects on oxidative stress and anchorage-independent growth. Similarly, Mitsui-7 did not significantly alter migratory behavior compared with control cells.

Collectively, these results indicate that chronic exposure to selected MWCNTs promotes migratory behavior in BEAS-2B cells, whereas migration is not uniformly induced across all materials. This suggests that MWCNT-induced migration represents a material-specific malignant phenotype rather than a general class effect and does not consistently parallel other transformation-related endpoints such as anchorage-independent growth.

Figure 6. Effects of long-term MWCNT exposure on migratory capacity of BEAS-2B cells. The BEAS-2B cells were chronically exposed to MWCNTs for 4 weeks and subsequently evaluated for cell migration using a wound healing assay. Exposed cells were seeded into 12-well plates at a density of 5 × 10⁵ cells per well. A linear scratch was introduced, and wound closure was monitored at 0 and 48 h. (a) Representative images of wound closure at 0 and 48 h following exposure to MWCNTs at concentrations of 1 and 10 μg/mL. (b) Quantitative analysis of migration expressed as the percentage of remaining wound area relative to the untreated control. Higher values indicate increased migratory capacity due to enhanced wound closure. Data represent the mean ± standard deviation (SD) of three independent experiments. Statistical significance compared with the control group is indicated as * p < 0.05.

Figure 6. Effects of long-term MWCNT exposure on migratory capacity of BEAS-2B cells. The BEAS-2B cells were chronically exposed to MWCNTs for 4 weeks and subsequently evaluated for cell migration using a wound healing assay. Exposed cells were seeded into 12-well plates at a density of 5 × 10⁵ cells per well. A linear scratch was introduced, and wound closure was monitored at 0 and 48 h. (a) Representative images of wound closure at 0 and 48 h following exposure to MWCNTs at concentrations of 1 and 10 μg/mL. (b) Quantitative analysis of migration expressed as the percentage of remaining wound area relative to the untreated control. Higher values indicate increased migratory capacity due to enhanced wound closure. Data represent the mean ± standard deviation (SD) of three independent experiments. Statistical significance compared with the control group is indicated as * p < 0.05.

3.5.2. Cell Invasion Assessed by the Transwell–Matrigel Assay

Cell invasion, defined as the three-dimensional migration of cells through the extracellular matrix (ECM), represents a critical feature of malignant progression [24]. The invasive potential of BEAS-2B cells following chronic exposure to MWCNTs was evaluated using a Matrigel-coated Transwell invasion assay. As shown in Figure 7, invasive activity was differentially modulated depending on the nanomaterial type.

Among the tested materials, MWCNT 1 induced a significant increase in invasion at 1 µg/mL (137% of control), while Mitsui-7 elicited the most pronounced pro-invasive response, showing significant increases at both 1 µg/mL (158%) and 10 µg/mL (115%). MWCNT 2 produced only modest but statistically significant elevations in invasion (approximately 110–113%), whereas MWCNT 3 and MWCNT 4 did not induce significant changes relative to the control at either concentration.

Notably, the induction of invasive behavior did not uniformly correlate with exposure concentration, as certain materials exhibited stronger effects at lower doses. These findings indicate that MWCNT-induced invasion is a material-specific phenotype rather than a uniform class effect and may represent a later-stage or context-dependent transformation-related response. In combination with migration and anchorage-independent growth data, the results suggest that prolonged exposure to selected MWCNTs can promote distinct malignant traits in bronchial epithelial cells, while other materials preferentially induce alternative transformation-related endpoints.

Figure 7. Effects of long-term MWCNT exposure on the invasive potential of BEAS-2B cells. The invasive capacity of BEAS-2B cells following chronic exposure to MWCNTs for 4 weeks was evaluated using a Transwell Matrigel invasion assay. Exposed cells were seeded into Matrigel-coated Transwell inserts (8-μm pore size) and allowed to invade toward a serum-containing chemoattractant for 48 h. (a) Representative images of invaded cells following exposure to MWCNTs at concentrations of 1 and 10 μg/mL. Invaded cells were stained with crystal violet. (b) Quantitative analysis of cell invasion expressed as a percentage relative to untreated control cells. Data represent the mean ± standard deviation (SD) of three independent experiments. Statistical significance compared with the control group is indicated as ** p < 0.01.

Figure 7. Effects of long-term MWCNT exposure on the invasive potential of BEAS-2B cells. The invasive capacity of BEAS-2B cells following chronic exposure to MWCNTs for 4 weeks was evaluated using a Transwell Matrigel invasion assay. Exposed cells were seeded into Matrigel-coated Transwell inserts (8-μm pore size) and allowed to invade toward a serum-containing chemoattractant for 48 h. (a) Representative images of invaded cells following exposure to MWCNTs at concentrations of 1 and 10 μg/mL. Invaded cells were stained with crystal violet. (b) Quantitative analysis of cell invasion expressed as a percentage relative to untreated control cells. Data represent the mean ± standard deviation (SD) of three independent experiments. Statistical significance compared with the control group is indicated as ** p < 0.01.

3.6. Tumorigenic Effects of BEAS-2B Cells Derived from Anchorage-Independent Colonies

3.6.1. Proliferation of Cells Harvested from Anchorage-Independent Colonies

Cells that acquire malignant characteristics frequently display dysregulated proliferative control, resulting in sustained cell growth [25]. In this study, BEAS-2B cells were chronically exposed to MWCNTs for 4 weeks, after which colonies formed under anchorage-independent conditions were harvested and further evaluated for proliferative capacity under adherent conditions. These assays were designed to assess the stability and persistence of transformed phenotypes in colony-derived cells following chronic MWCNT exposure, and to confirm that these cells retained sufficient proliferative capacity under anchorage-dependent conditions to support subsequent xenograft implantation.

As shown in Figure 8a, cells derived from anchorage-independent colonies exhibited markedly increased proliferation rates compared with parental BEAS-2B cells. Notably, cells derived from Mitsui-7-induced colonies showed the most pronounced increase, reaching approximately 700% of the control level. Cells derived from other MWCNT-exposed groups also demonstrated significantly elevated proliferation. These results indicate that BEAS-2B cells selected through anchorage-independent growth acquire a stable proliferative advantage following chronic MWCNT exposure.

3.6.2. Anchorage-Dependent Colony Formation of Cells Harvested from Anchorage-Independent Colonies

To further assess the clonogenic potential of transformed cells, colony-derived BEAS-2B cells were subjected to an anchorage-dependent colony formation assay. Cells harvested from soft agar colonies following 4 weeks of MWCNT exposure were reseeded onto standard tissue culture plates and cultured under adherent conditions. As shown in Figure 8b, transformed cells exhibited a substantial increase in anchorage-dependent colony formation compared with the control group. Quantitative analysis revealed marked increases in colony formation for MWCNT 1 (1 µg/mL, approximately 150% of control), MWCNT 2 (1 µg/mL, approximately 1000%; 10 µg/mL, approximately 900%), MWCNT 3 (1 µg/mL, approximately 3400%), MWCNT 4 (1 µg/mL, approximately 4025%; 10 µg/mL, approximately 2200%), and Mitsui-7 (1 µg/mL, approximately 260%).

Notably, although Mitsui-7-derived cells formed fewer colonies compared with several domestically manufactured MWCNTs, the colonies formed by Mitsui-7 were visibly larger in size (Figure 8b). This qualitative observation is consistent with the markedly enhanced proliferative capacity of Mitsui-7-derived cells observed in subsequent proliferation assays (Figure 8a), suggesting that Mitsui-7 exposure preferentially promotes robust clonal expansion rather than increasing colony number alone.

Collectively, these findings indicate that chronic exposure to different MWCNTs induces distinct clonogenic growth patterns, with some materials favoring increased colony number and others promoting enhanced colony growth and proliferative dominance. Such material-specific differences in anchorage-dependent clonogenic behavior further support the heterogeneous malignant transformation potential among MWCNTs.

3.6.3. Tumorigenic Potential of Transformed BEAS-2B Cells in a Xenograft Model

The in vivo tumorigenic potential of transformed BEAS-2B cells was evaluated using a xenograft mouse model. Colony-derived cells harvested from the soft agar assay were subcutaneously injected into athymic nude mice, and tumor growth was monitored for 15 days (Figure 9a).

As shown in Figure 9b–d, all MWCNT-treated groups developed significantly larger tumors compared with the control group, in which no palpable tumors were observed. The most pronounced tumorigenic response was observed in mice injected with MWCNT 3-derived cells, which formed the largest tumors (mean tumor volume > 1500 mm³; mean weight approximately 0.76 g). This was followed by MWCNT 2-derived cells (approximately 1000 mm³ and 0.70 g) and MWCNT 4-derived cells (approximately 870 mm³ and 0.66 g).

Mitsui-7-derived cells induced moderate but significant tumor growth (approximately 500 mm³ and 0.38 g), whereas MWCNT 1-derived cells resulted in smaller yet still significant tumors (approximately 420 mm³ and 0.35 g).

Collectively, these results demonstrate that chronic exposure to MWCNTs induces stable tumorigenic phenotypes in BEAS-2B cells, enabling robust tumor formation in vivo following selection through anchorage-independent growth.

Figure 9. Xenograft tumorigenicity of BEAS-2B cells derived from soft agar colonies. BEAS-2B cells transformed following chronic exposure to individual MWCNTs were harvested from soft agar colonies, and 1 × 10⁶ cells were subcutaneously injected into BALB/c nude mice. (a) Schematic illustration of the xenograft tumorigenicity assay. (b) Representative images of excised tumor xenografts on day 15 post-injection, with corresponding tumor weights (g) indicated for each sample. (c) Quantitative analysis of tumor volume. Data are presented as the mean ± SD from three independent experiments. (d) Quantitative analysis of tumor weight. Data are presented as the median ± SD from three independent experiments. Mice injected with BEAS-2B cells transformed by MWCNT 2, MWCNT 3, or Mitsui-7 developed significantly larger tumors compared with the control group. Statistical significance compared with the control group is indicated as ** p < 0.01.

Figure 9. Xenograft tumorigenicity of BEAS-2B cells derived from soft agar colonies. BEAS-2B cells transformed following chronic exposure to individual MWCNTs were harvested from soft agar colonies, and 1 × 10⁶ cells were subcutaneously injected into BALB/c nude mice. (a) Schematic illustration of the xenograft tumorigenicity assay. (b) Representative images of excised tumor xenografts on day 15 post-injection, with corresponding tumor weights (g) indicated for each sample. (c) Quantitative analysis of tumor volume. Data are presented as the mean ± SD from three independent experiments. (d) Quantitative analysis of tumor weight. Data are presented as the median ± SD from three independent experiments. Mice injected with BEAS-2B cells transformed by MWCNT 2, MWCNT 3, or Mitsui-7 developed significantly larger tumors compared with the control group. Statistical significance compared with the control group is indicated as ** p < 0.01.

4. Discussion

In the present study, we applied an adverse outcome pathway (AOP)—oriented integrated testing strategy to systematically evaluate the carcinogenic potential of domestically manufactured MWCNTs in comparison with Mitsui-7, a reference MWCNT classified as possibly carcinogenic to humans. As outlined in the AOP-based framework (Figure 1), enhancement of anchorage-independent growth and xenograft tumorigenicity represents critical downstream key events along the progression toward adverse carcinogenic outcomes. By aligning a series of in vitro and in vivo assays with successive key events of carcinogenesis, we demonstrated that chronic exposure to specific MWCNTs induces progressive malignant phenotypes in human bronchial epithelial BEAS-2B cells, ultimately resulting in tumor formation in a xenograft model.

It is important to acknowledge that BEAS-2B cells are SV40 large T antigen–immortalized human bronchial epithelial cells and therefore exhibit an increased susceptibility to transformation compared with primary cells [16,26]. Nevertheless, BEAS-2B cells are non-malignant and have been extensively used as experimental models for investigating malignant transformation and long-term exposure-induced phenotypic changes [27,28,29]. Immortalized bronchial epithelial cell lines are particularly valuable for studying chronic and repeated exposure scenarios that are not technically feasible using primary human airway epithelial cells. Consequently, BEAS-2B cells have been widely employed as an in vitro screening models for assessing the pulmonary toxicity and carcinogenic potential of chemical and biological agents [27,30,31,32]. Within this context, the present findings should be interpreted as evidence of material-specific transforming and tumorigenic potential under defined experimental conditions, rather than as direct evidence of carcinogenicity relevant to human exposure or population-level cancer risk.

A strong concordance was observed between cytotoxicity and intracellular ROS generation across the tested MWCNTs, indicating that oxidative stress represents a critical early cellular responses following acute exposure. Oxidative stress has been widely implicated in carcinogenic processes, as excessive ROS has been reported in various experimental systems to contribute to oxidative DNA damage, genomic instability, and dysregulation of redox-sensitive signaling pathways [21,33]. However, genotoxic endpoints were not directly assessed in the present study, and therefore ROS induction is discussed within a non-genotoxic carcinogenesis framework.

Although EDS analysis confirmed the dominant carbon composition of the tested MWCNTs, this method has limited sensitivity for detecting trace levels of residual metal catalysts such as Fe, Ni, or Co. Accordingly, a minor contribution of metal impurities to oxidative stress cannot be completely excluded. Importantly, however, the material-dependent oxidative stress responses observed in this study were more closely associated with differences in aggregation state and fiber morphology than with elemental composition, supporting a primary role of intrinsic physicochemical properties in driving early cellular stress responses.

While H&E staining provided supportive evidence of MWCNT association with exposed BEAS-2B cells (Figure S2), higher-resolution imaging approaches such as SEM or TEM will be required in future studies to definitively resolve aggregation-dependent membrane interactions, internalization, and subcellular localization.

Notably, MWCNT 3, which exhibited the highest degree of dense aggregation and fiber entanglement based on SEM analysis, induced the most pronounced cytotoxicity and ROS production. Previous studies have reported that highly aggregated and entangled high-aspect-ratio carbon nanomaterials can promote persistent oxidative stress through impaired cellular clearance, prolonged intracellular retention, and frustrated phagocytic responses [34,35]. Importantly, the elevated ROS levels induced by MWCNT 3 did not primarily translate into sustained cytotoxicity or irreversible cell death. Instead, following short-term exposure and subsequent recovery periods, colony formation efficiency and crystal violet assays revealed a distinct response pattern characterized by enhanced cell survival, proliferation and colony formation rather than cytotoxic suppression. This response profile is consistent with promotion-stage carcinogenic processes, in which sustained oxidative stress acts as a non-genotoxic driver of cell proliferation, clonal expansion and phenotypic selection rather than direct cell elimination [36,37]. Recent advances in non-genotoxic carcinogenesis research further emphasize that enhanced cell proliferation represents a central key event linking early cellular stress responses to tumor development, particularly in the absence of experimentally confirmed genotoxic damage [38]. These observations indicate that differences in aggregation behavior among the tested MWCNTs reflect inherent physicochemical characteristics of the starting materials rather than dispersion-induced artifacts. This distinction supports a causal relationship between intrinsic aggregation-related properties and the differential biological responses observed in this study and provides a mechanistic basis for hazard differentiation among MWCNTs within an AOP-oriented framework.

Within non-genotoxic carcinogenesis AOPs, oxidative stress is positioned upstream of increased cell proliferation as a key event bridging early cellular perturbations to downstream tumorigenic outcomes [37,38]. In chronic exposure models, clonal expansion can arise either from survival-based selection under sub-lethal stress or from promotion-stage carcinogenic mechanisms driven by sustained proliferative signaling.

In the present study, although exposure to certain MWCNTs approached cytotoxic thresholds in short-term assays, prolonged exposure did not result in sustained cytotoxicity. Instead, the acquisition of anchorage-independent growth, stable clonogenic capacity upon reseeding, and robust tumor formation in xenograft models suggests that the observed clonal expansion reflects stable transforming and tumorigenic phenotypes rather than transient survival-based selection alone. Consistent with this framework, MWCNT-induced oxidative stress was associated with enhanced clonal expansion and malignant phenotypes rather than sustained cytotoxicity.

As illustrated in the AOP-based integrated testing strategy (Figure 1), oxidative stress represents an early key event that can propagate downstream through increased cell proliferation, anchorage-independent growth, and ultimately tumor formation. The xenograft assay employed in this study evaluates the tumorigenic potential of transformation-derived, colony-selected cells rather than de novo carcinogenesis occurring in vivo. Accordingly, the in vivo findings should be interpreted as confirmation that cells acquiring anchorage-independent growth following chronic MWCNT exposure can sustain malignant growth under permissive conditions, rather than as direct evidence of carcinogenic initiation in animals or humans. This sequential pattern supports an AOP-consistent continuum linking ROS induction to clonal expansion and tumorigenicity rather than a simple cytotoxic cascade.

The MWCNTs examined in this study represent established reference materials whose detailed physicochemical characteristics have been previously defined [3]. Accordingly, the present work focused on linking these known structural features to functional carcinogenic outcomes within an AOP-oriented framework, rather than re-establishing primary material characterization.

The present findings further suggest that distinct physicochemical features of MWCNTs may drive carcinogenic outcomes through distinct mechanistic pathways. In particular, the carcinogenic profile of MWCNT 3 differed markedly from that of Mitsui-7. While Mitsui-7 is characterized by a relatively straight and rigid fiber morphology, MWCNT 3 exhibited dense aggregation and extensive entanglement. These morphological differences were associated with divergent biological response patterns. Rigid and straight MWCNTs such as Mitsui-7 have been reported to induce carcinogenic outcomes primarily through physical interactions with cellular structures and persistent inflammation, resembling asbestos-like pathogenicity in vivo [4,39]. In contrast, the present study demonstrates that highly aggregated MWCNTs can promote malignant transformation through a proliferation-driven pathway associated with sustained oxidative stress and clonal expansion.

In this context, MWCNT 3 consistently induced higher intracellular ROS levels, enhanced colony formation efficiency, pronounced anchorage-independent growth, and robust tumor formation in xenograft models, supporting a strong functional association between aggregation-induced oxidative stress and tumorigenic potential. These findings suggest that aggregation-dominant MWCNTs may preferentially activate promotion-stage carcinogenic mechanisms characterized by enhanced survival, clonal expansion, and sustained proliferative signaling. In contrast, rigidity-dominant MWCNTs such as Mitsui-7 have been reported to exert carcinogenic effects primarily through chronic physical injury and inflammation. Although these pathways appear mechanistically distinct, both ultimately converge on tumorigenic outcomes, underscoring the importance for material-specific hazard evaluation rather than reliance on a single morphological descriptor.

Interestingly, malignant phenotypes did not progress uniformly across all tested MWCNTs. Chronic exposure to MWCNT 3 and MWCNT 4 did not result in significant changes in either migratory or invasive capacity, despite inducing robust anchorage-independent growth and pronounced tumor formation in the xenograft model. This finding indicates that enhanced tumorigenicity can occur in the absence of overt migration or invasion phenotypes at the cellular level. The xenograft model employed in this study was designed to assess tumor-forming capacity at the primary site following anchorage-independent selection, rather than invasive growth or metastatic dissemination. Therefore, the absence of a direct correspondence between in vitro migration/invasion phenotypes and in vivo tumor growth does not preclude tumorigenic potential but rather reflects the stage-specific acquisition of malignant traits within an AOP-consistent framework. Evaluation of invasive growth and metastasis will require more complex in vivo models, including orthotopic or long-term metastatic assays.

Overall, these findings support an AOP-consistent framework in which increased proliferation and anchorage-independence growth represent key downstream events linking early cellular stress responses to tumorigenic outcomes, while migration and invasion constitute complementary but non-essential hallmarks that may be acquired during later stages of malignancy. By integrating SEM-based morphological characterization with functional cellular and in vivo endpoints, this study provides mechanistic insight into how aggregation density and fiber entanglement translate into carcinogenic outcomes along an AOP continuum. Importantly, the concentrations used in this study (1–20 μg/mL) exceed predicted levels of human respiratory tract exposure, even under occupational settings. Accordingly, these findings should be interpreted as evidence of intrinsic carcinogenic potential under experimental conditions rather than as a basis for direct quantitative risk assessment in real-world exposure scenarios.

Several limitations of present study should be acknowledged. While the applied AOP-oriented integrated testing strategy enabled systematic evaluation of non-genotoxic carcinogenic key events, detailed molecular pathway analyses were beyond the scope of this work and will be required in future studies to elucidate the stress-responsive signaling mechanisms underlying aggregation-driven transformation. In addition, although quantitative dispersion metrics were not included, all tested MWCNTs exhibited comparable sedimentation behavior under identical exposure conditions, and no correlation was observed between sedimentation and biological responses, suggesting that dispersion artifacts are unlikely to account for the material-dependent effects observed.

5. Conclusions

In conclusion, this study applied an AOP-oriented ITS to systematically evaluate the carcinogenic potential of domestically manufactured MWCNTs in comparison with Mitsui-7, a reference MWCNT classified as possibly carcinogenic to humans. By integrating physicochemical characterization with a sequence of functionally anchored in vitro assays and confirmatory in vivo xenograft outcomes, this approach enabled a coherent assessment of key events relevant to non-genotoxic carcinogenesis following long-term exposure.

Within the AOP framework, aggregation-driven oxidative stress emerged as a critical early key event that propagated downstream to increased cell proliferation, clonal expansion, anchorage-independent growth, and tumorigenic potential, corresponding to promotion and early progression stages of carcinogenesis. Importantly, carcinogenic responses did not progress uniformly across the tested materials, demonstrating that MWCNT-associated carcinogenic potential is strongly influenced by intrinsic physicochemical heterogeneity rather than representing a uniform class effect.

Collectively, these findings demonstrate the applicability and added value of an AOP-aligned ITS for identifying and differentiating non-genotoxic carcinogenic hazards of nanomaterials. The strategy presented here provides a mechanistically informed and regulatory-relevant framework that may support future hazard identification, material grouping, and weight-of-evidence-based decision-making within nanosafety assessment and OECD-aligned testing paradigms for emerging carbon-based nanomaterials.