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 (t_1/2) for both particle sizes was, in general, longer in male rats than in male mice. In male rats, the t_1/2 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.

Synonyms: 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

Contents

• Foreword
• Collaborators
• Contributors
• Peer Review
• Publication Details
• Acknowledgements
• Preface
• Introduction
  • Chemical and Physical Properties
  • Production, Use, and Human Exposure
  • Regulatory Status
  • Absorption, Distribution, Metabolism, and Excretion
  • Toxicity
  • Reproductive and Developmental Toxicity
  • Carcinogenicity
  • Genetic Toxicity
  • Study Rationale
• Materials and Methods
  • Procurement and Characterization of Bulk Fullerene C₆₀
  • Aerosol Generation and Exposure System for the Micro-C₆₀ Studies
  • Aerosol Generation and Exposure System for the Nano-C₆₀ Studies
  • Aerosol Concentration Monitoring for the Micro-C₆₀ and Nano-C₆₀ Studies
  • Carousel Atmosphere Characterization for the Micro-C₆₀ and Nano-C₆₀ Studies
  • Animal Source
  • Animal Welfare
  • Three-month Studies
  • Statistical Methods
  • Quality Assurance Methods
  • Genetic Toxicology – Peripheral Blood Micronucleus Test
• Results
  • Data Availability
  • Three-month Studies in Rats
  • Three-month Studies in Mice
  • Genetic Toxicology
• Discussion
• References
• Appendix A. Reproductive Tissue Evaluations and Estrous Cycle Characterization
• Appendix B. Tissue Burden Results
• Appendix C. Immunotoxicity Results
• Appendix D. Genetic Toxicology
• Appendix E. Chemical Characterization and Generation of Carousel Concentrations
• Appendix F. Ingredients, Nutrient Composition, and Contaminant Levels in NTP-2000 Rat and Mouse Ration
• Appendix G. Sentinel Animal Program
• Appendix H. Supplemental Data