National Toxicology Program

National Toxicology Program

Abstract from Reports I20407B and I20710B on C60 Fullerene

Range-Finding Report on the Immunotoxicity of C60 Fullerene in Female B6C3F1 Mice (CAS No. 99685-96-8)

Report Date: November 2012

The following abstract presents results of a study conducted by a contract laboratory for the National Toxicology Program. The findings have not been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by NTP in March 2009. The findings and conclusions for this study should not be construed to represent the views of the NTP or the U.S. Government.


The in-life phase of the study was conducted at Battelle Northwest (Battelle), Richland, WA, under Battelle Protocol Nos. 20407-02 and 20701-02 as a part of National Toxicology Program Contract No. N01-ES-45517. At the request of the Project Officer, an immunotoxicological evaluation was conducted at Virginia Commonwealth University, Richmond, Virginia, under NTP Contract No. N01-ES-55538. The Study Director for the immunotoxicological evaluation phase of the study was Kimber L. White, Jr., Ph.D., while Tai L. Guo, Ph.D., DABT, served as the Assistant Study Director.

The objective of the study was to determine the potential effects of C60 Fullerene (Chemical Abstracts Service [CAS] #99685-96-8) on the immune system of female B6C3F1 mice, when administered for 13-weeks by nose only inhalation. In evaluating the effect of C60 on the immune response, two particle sizes were evaluated. The larger particle size, 1.0 µm diameter C60, was evaluated at three concentrations (2, 15, and 30 mg/m3). The smaller particle size, 0.05 µm diameter C60, was evaluated at two concentrations (0.5 and 2 mg/m3). These smaller particles meet the criteria of being "nanoparticles," which are defined as particles with a length in at least one dimension ranging between 1 and 100 nm. The study consisted of two cohorts. Cohort 1 animals were immunized with sheep erythrocytes, a T-dependent antigen. After immunization, the animals in Cohort 1 were evaluated for their antigen specific antibody responses to sRBC by evaluating the antibody-forming cell response and serum anti-sRBC immunoglobulin M antibody levels. Animals in Cohort 2 were not immunized and were used for the following immunotoxicological assays: splenocyte phenotyping, mixed leukocyte response, anti-CD3 mediated proliferation assay, the natural killer cell assay, mediastinal lymph node cell differentials, bronchoalveolar lavage cell differentials, and BAL fluid cytokine levels. On the day of study termination, spleens, mediastinal lymph nodes, and BAL cells were placed in tubes containing media, placed on crushed ice, and shipped to VCU in Richmond, VA, for immunoassay evaluations on the following day. The collected serum and BAL fluid samples were frozen and shipped separately on dry ice to VCU, where they were maintained at -70°C until evaluated. Upon receipt, it was determined that the mediastinal lymph nodes and BAL cells were unusable for immunotoxicological evaluation because of contamination with erythrocytes and other tissues. No other significant protocol or standard operating procedure deviations occurred during the study that affected the quality of the data and the ability to interpret the data with respect to the immunotoxicology of C60.

For parameters evaluated for the 1.0 µm C60 (Battelle Protocol 20701-02), no statistically significant effects were observed in the AFC assay, serum IgM antibody levels to sRBC, splenic phenotypic analysis, anti-CD3 mediated proliferation, or the NK cell assay. The MLR was decreased at the 2 and 15 mg/m3 exposure levels, but not at the 30 mg/m3 dose. No differences from control were observed for 13 of the 14 cytokines evaluated in the BAL fluid. The only cytokine affected was MIP-1α, which was significantly increased at the 15 and 30 mg/m3 exposure levels in both the neat and concentrated samples. In the concentrated BAL fluid samples, MIP-1α levels in the 15 and 30 mg/m3 exposure groups were 3,024% and 31,878% higher than those of the control group, respectively.

For parameters evaluated for the 0.05 µm (nanoparticle) C60 (Battelle Protocol No. 20407-02), similar to the results observed with the larger C60 particles, the 0.05 µm C60 did not significantly affect the AFC response, serum anti-sRBC IgM antibody titers, spleen cell phenotypes, the MLR, or NK cell activity, when compared to the control group. The anti-CD3 mediated proliferative response was decreased, reaching the level of statistical significance at the 2 mg/m3 exposure level. Spleen cell numbers in the Cohort 2 animals (i.e., those not immunized with sRBC) were significantly decreased at the 2 mg/m3 dose. No differences were observed in the levels of any of the fourteen BAL fluid cytokines evaluated when compared to the control group.

In conclusion, under the experimental conditions of this study, C60 had minimal effects on the various components of the systemic immune system, including innate immunity, humoral immunity, and cell-mediated immunity when administered by nose only inhalation for 13 weeks to female B6C3F1 mice. Furthermore, the size of the C60 particle administered did not affect the results, since neither the micron-sized particles (1.0 µm) nor the nanoparticles (0.05 µm) produced consistent significant effects on the systemic immune system. The only notable difference between the two particle sizes was the effect on MIP-1α levels in the BAL fluid, where mice exposed to 1.0 µm C60 demonstrated a significant increase in this cytokine, while those exposed to 0.05 µm C60 demonstrated no such effect.

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