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https://ntp.niehs.nih.gov/go/tox087abs

Abstract for TOX-87

Toxicity Studies of Fullerene C60 (1 μm and 50 nm) Administered by Nose-only Inhalation to Wistar Han [Crl:WI (Han)] Rats and B6C3F1/N Mice

CASRN: 99685-96-8
Chemical Formula: C60
Molecular Weight: 720.6
Synonyms/Common Names: Buckminsterfullerene; Buckminsterfullerene (C60); Buckyball, C60 fullerenecarbon (C60); Carbon cluster (C60); Carbon C60 fullerene; Carbon (C60) mol.; Carbon, mol. (C60); Follene-60; Footballene; Footballene (C60); [60]fullerene; [5,6]fullerene C60; Fullerene-60: Fullerene-C60; Fullerene C60 cluster; Icosahedral C60; Soccerballene
Report Date: July 2020

FULL REPORT PDF

Abstract

Fullerene C60 (C60), a primary allotrope of carbon, is used in a variety of consumer applications including microelectronics, photovoltaics, batteries and fuel cells, and water treatment methods. Human exposure to engineered C60 due to industrial applications may occur via inhalation, oral, dermal, or parenteral routes. In these toxicity and tissue burden studies, male and female Wistar Han rats and B6C3F1/N mice were exposed to fullerene C60 (at least 95% pure) via nose-only inhalation for 3 months. Two different C60 fullerene aggregate sizes, 1 µm diameter (micro-C60) and 50 nm diameter (nano-C60) were studied to assess the potential for differential effects based on particle size.

Groups of 10 male and 10 female core study rats and mice were exposed to atmospheres of the two fullerene C60 particle sizes via nose-only inhalation at concentrations of 0, 2, 15, or 30 mg/m3 (micro-C60 studies) or 0, 0.5, or 2 mg/m3 (nano-C60 studies), 3 hours per day, 5 days per week for 13 weeks. Additional groups of rats and mice were analyzed for tissue burden and studied for clearance studies and immunotoxicity.

Lung tissue weights were significantly increased at multiple timepoints in male rats and mice exposed to micro- or nano-C60 particles. Lung tissue burden increased with exposure concentration following exposure to either micro- or nano-C60 particles and was greater in rats and mice exposed to the same concentration (2 mg/m3) of nano C60 relative to micro-C60 particles. The calculated lung clearance half-life (t1/2) for both particle sizes was, in general, longer in male rats than in male mice. In male rats, the t1/2 of micro-C60 was shorter than that of nano-C60 particles at the same mass-based exposure concentration (2 mg/m3).

In the micro-C60 study in rats no exposure-related effects on body weights or clinical pathology were found in male or female rats. Liver weights of 30 mg/m3 males were significantly greater than those of the control males. Histologically, exposure-related increases in the lung (chronic active inflammation and histiocyte infiltration) of both male and female rats occurred. Exposure-related increases in pigmentation (presumably test article) were seen in the lung and the bronchial (male) and mediastinal (females) lymph nodes. Micro-C60 exposure exhibited the potential to be a reproductive toxicant in male rats on the basis of decreased sperm motility and low incidences of histopathological findings in the testes (germinal epithelium degeneration). In the micro-C60 study in mice, mean body weights of exposed groups of males and females were similar to those of the control groups. Lung weights of 30 mg/m3 exposed females were significantly higher than those of the control females. There were no exposure-related effects on hematology in male or female mice. Histologically, exposure-related lesions occurred in the lung (chronic active inflammation and histiocyte infiltration) and larynx (squamous metaplasia). Exposure-related increases in pigmentation were seen in the lung and bronchial lymph nodes (males). Micro-C60 exposure also exhibited the potential to be a reproductive toxicant in female mice on the basis of increased probability of extended estrus and in male mice on the basis of decreased sperm motility.

In the nano-C60 study in rats, no effects were observed on body weights, clinical pathology, or organ weights, and no exposure-related increases were found in chronic active inflammation and histiocyte infiltration in the lung. Exposure-related increases in pigmentation were seen in the lung (females) and bronchial lymph nodes. Low incidences of germinal epithelium degeneration in the testis and hypospermia were observed in male rats in the 2 mg/m3 group compared to none in control males. Sperm motility was significantly reduced in all exposed groups. Nano C60 exposure exhibited the potential to be a reproductive toxicant in male rats, but not in male mice, on the basis of lower sperm motility and low incidences of histopathological findings in the testis (germinal epithelium degeneration) and epididymis (hypospermia). In the nano-C60 study in mice, there were no exposure-related effects on body weights, organ weights, and clinical pathology and no exposure-related increase in chronic active inflammation as seen with micro-C60. Exposure-related increases in histiocyte infiltration were seen in the lungs of male mice. Exposure-related increases in pigmentation were seen in the lung and bronchial lymph nodes (males). Nano-C60 exposure exhibited the potential to be a reproductive toxicant in female mice on the basis of increased probability of extended estrus compared to control females.

Micro- and nano-C60 exposure had minimal effects on the components of the immune system examined, including innate, humoral, and cell-mediated immunity, in female rats and mice. Bronchoalveolar lavage fluid (BALF) macrophages from female rats and mice exposed to nano C60 and micro C60 contained intracytoplasmic, brown to black, granular to globular pigment. Mice exposed to micro-C60 demonstrated a significant increase in MIP 1α levels in the BALF, while those exposed to nano-C60 demonstrated no such effect. Alterations in BALF cell phenotype were observed in female Wistar Han rats at all exposure levels. Levels of specific cytokines in the BALF were altered following exposure to micro- or nano-C60 in both rats and mice. Shifts in spleen cell phenotype were noted in B6C3F1/N female mice exposed to either micro- (15 and 30 mg/m3) or nano- (0.5 and 2 mg/m3) C60.

Neither micro- nor nano-C60 increased the frequencies of micronucleated reticulocytes or erythrocytes in peripheral blood of male or female Wistar Han rats or B6C3F1/N mice. Neither size particle affected the percentage of reticulocytes in peripheral blood, suggesting no exposure-related bone marrow toxicity.

In conclusion, C60 fullerene nose-only inhalation in both rats and mice for 13 weeks had little effect on systemic immune function. Locally, lung inflammatory responses were observed, primarily at the higher exposure concentrations. Inflammatory effects of nano-C60 exposure were, in general, more severe than micro-C60 exposure at the same mass-based atmospheric exposure concentrations.

National Toxicology Program (NTP). 2020. NTP technical report on the toxicity studies of fullerene C60 (1 µm and 50 nm) (CASRN 99685-96-8) administered by nose-only inhalation to Wistar Han [Crl:WI (Han)] rats and B6C3F1/N mice. Research Triangle Park, NC: National Toxicology Program. Toxicity Report 87. https://doi.org/10.22427/NTP-TOX-87

Studies

Summary of Toxicologically Relevant Findings in Rats and Mice in the Three-month Nose-only Inhalation Study of Fullerene Micro-C60 (1 µm)
  Male
Wistar Han Rats
Female
Wistar Han Rats
Male
B6C3F1/N Mice
Female
B6C3F1/N Mice
Concentrations in air 0, 2, 15, or 30 mg/m3 0, 2, 15, or 30 mg/m3 0, 2, 15, or 30 mg/m3 0, 2, 15, or 30 mg/m3
Survival rates 10/10, 10/10, 10/10, 10/10 10/10, 9/10, 10/10, 10/10 10/10, 9/10, 10/10, 10/10 10/10, 10/10, 10/10, 10/10
Body weights Exposed groups similar to the control group Exposed groups similar to the control group Exposed groups similar to the control group Exposed groups similar to the control group
Lung burden
µg C60/total lung[a] 439; 2,182; 5,749 234; 1,763; 4,658 38; 209; 595 36; 190; 560
t1/2 (days) 27, 69, 55 Not assessed 16, 18, 68 Not assessed
Organ weights ↑ Liver (absolute and relative)[b] None None ↑ Lung (absolute and relative)
Clinical pathology None None None None
Immunotoxicity Not assessed BALF: pigmented macrophages in all exposed groups; ↑ % neutrophils at 30 mg/m3: ↑ % lymphocytes at 15 and 30 mg/m3: ↑IL-1 at 15 and 30 mg/m3: ↑MCP-1 at 30 mg/m3 Not assessed BALF: pigmented macrophages in exposed groups. ↑ % neutrophils and ↑ % lymphocytes at 15 and 30 mg/m3: ↑MIP-1α at 15 and 30 mg/m3
Reproductive toxicity ↓ Sperm motility Not assessed ↓ Sperm motility ↑ Probability of extended estrus
Nonneoplastic effects Lymph node, bronchial: pigmentation (0/7, 1/5, 3/6, 7/8)

Lung: infiltration cellular, histiocyte (1/10, 1/10, 8/9, 10/10); inflammation, chronic active (1/10, 4/10, 8/9, 10/10); pigmentation (0/10, 1/10, 8/9, 10/10)

Testes: germinal epithelium (0/10, 1/10, 3/10, 1/10)
Lymph node, mediastinal: pigmentation (3/8, 9/10, 7/8, 7/9)

Lung: infiltration cellular, histiocyte (1/10, 1/10, 8/10, 10/10); inflammation, chronic active (0/10, 0/10, 1/10, 10/10); pigmentation (0/10, 0/10, 8/10, 10/10)
Lymph node, bronchial: pigmentation (2/6, 1/4, 2/5, 9/9)

Larynx: metaplasia, squamous (1/10, 0/9, 5/10, 9/10)

Lung: infiltration cellular, histiocyte (0/10, 0/10, 10/10, 10/10); inflammation, chronic active (0/10, 0/10, 10/10, 10/10); pigmentation (0/10, 0/10, 10/10, 10/10)
Larynx: metaplasia, squamous (0/10, 1/8, 6/10, 9/10)

Lung: infiltration cellular, histiocyte (0/10, 0/10, 10/10, 10/10); inflammation, chronic active (0/10, 0/10, 3/10, 10/10); pigmentation (0/10, 0/10, 10/10, 10/10)
Genetic Toxicology
Assay Results
Micronucleated erythrocytes:
Peripheral blood in vivo:


Negative in males and female rats and mice

t1/2 = half-life
BALF = Bronchoalveolar lavage fluid
[a] = Postexposure day 0.
[b] =Relative to body weight

Summary of Toxicologically Relevant Findings Considered in Rats and Mice in the Three-month Nose-only Inhalation Study of Fullerene Nano-C60 (50 nm)
  Male
Wistar Han Rats
Female
Wistar Han Rats
Male
B6C3F1/N Mice
Female
B6C3F1/N Mice
Concentrations in air 0, 0.5, or 2 mg/m3 0, 0.5, or 2 mg/m3 0, 0.5, or 2 mg/m3 0, 0.5, or 2 mg/m3
Survival rates 10/10, 10/10, 10/10 10/10, 10/10, 10/10 10/10, 10/10, 10/10 10/10, 10/10, 10/10
Body weights Exposed groups similar to the control group Exposed groups similar to the control group Exposed groups similar to the control group Exposed groups similar to the control group
Lung burden
µg C60/total lung[a] 189, 581 128, 434 24, 115 26, 98
t1/2 (days) 38, 61 Not assessed 17, 15 Not assessed
Organ weights None None None None
Clinical pathology None None None None
Immunotoxicity Not assessed BALF: Pigmented macrophages in exposed groups Not assessed BALF: Pigmented macrophages in exposed groups
Reproductive toxicity ↓ Sperm motility Not assessed None ↑ Probability of extended estrus
Nonneoplastic effects Lymph node, bronchial: pigmentation (0/10, 3/9, 4/10)

Lymph node, mediastinal: pigmentation (1/10, 6/8, 2/10)

Testes: germinal epithelium: degeneration (0/10, 0/10, 3/10)
Lymph node, bronchial: pigmentation (0/10, 1/10, 5/10) Lymph node, mediastinal: pigmentation (0/8, 7/8, 9/10)

Lung: infiltration cellular, histiocyte (2/10, 1/10, 8/10); pigmentation (0/10, 0/10, 8/10)
Lymph node, bronchial: pigmentation (0/9, 2/7, 5/7)

Lung: infiltration cellular, histiocyte (1/10, 2/10, 7/10); pigmentation (0/10, 0/10, 7/10)
Lymph node, bronchial: pigmentation (2/10, 3/10, 4/10)

Lung: infiltration cellular, histiocyte (2/10,1/10, 5/10); pigmentation (0/10, 0/10, 5/10)
Genetic Toxicology
Assay Results
Micronucleated erythrocytes:
Peripheral blood in vivo:


Negative in males and female rats and mice

t1/2 = half-life
BALF = Bronchoalveolar lavage fluid
[a] = Postexposure day 0.
[b] =Relative to body weight