Range-Finding Report on the Immunotoxicity of C60 Fullerene in Female Wistar Han Rats (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 The findings have not been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by NTP in March 2009.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. 20407-01 and 20701-01 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 Wistar Han rats when administered for 13-weeks by nose only inhalation. In evaluating the effects 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 was used to conduct the Antibody-Forming Cell assay, for which the 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. Animals in Cohort 2 were not immunized and were used in the following immunotoxicological assays: splenocyte phenotyping, 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 BAL fluid was frozen and shipped separately on dry ice to VCU, where it was maintained at -70° C until evaluated. Upon receipt, it was determined that the mediastinal lymph nodes were unusable for immunotoxicological evaluation because of contamination with 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 the immunology parameters evaluated for the 1.0 µm C60 (Battelle Protocol 20701-01), no statistically significant effects were observed in any of the immunological assays evaluated, including the AFC assay, splenic phenotypic analysis, anti-CD3 mediated proliferation, NK cell activity, or BAL fluid cytokine levels. The only exceptions were a significant increase in BAL neutrophils at the 30 mg/m3 dose, significant decreases in BAL macrophages at all doses, and a significant increase in the level of MCP-1 in the BAL fluid, which was observed in the 30 mg/m3 exposure group in both the neat and the concentrated samples. An increase was observed in the 15 and 30 mg/m3 dose groups for IL-1 in the concentrated samples but not the neat samples. However, because IL-1 concentrations were near the limit of quantification and because they were lower in the concentrated samples than in the neat samples, these results are difficult to interpret.
For the immunology parameters evaluated for the 0.05 µm size (nanoparticle) C60 (Battelle Protocol No. 20407-01), similar to the results observed with the larger C60 particles, exposure to 0.05 µm C60 did not significantly affect any of the immunotoxicological parameters evaluated, as compared to the control group. The only exceptions were statistically significant decreases in BAL macrophages at all dose levels, and an increase in both spleen weight and spleen cell number at the 2 mg/m3 level in Cohort 1 (i.e., the immunized animals used in the plaque assay). Spleen weight and spleen cell numbers were not statistically different from the control group in the Cohort 2 group (i.e., the non-immunized animals).
In conclusion, under the experimental conditions of this study, C60 had minimal effects on the various components of the immune system examined, including innate immunity, humoral immunity, and cell-mediated immunity, when administered by nose only inhalation for 13 weeks to female Wistar Han rats. Furthermore, the size of the C60 particle administered did not affect the results, since neither the micron size particles (1.0 µm) nor the nanoparticles (0.05 µm) produced significant effects on the immune system. While exposure by the inhalation route to C60 did produce significant effects in the BAL fluid (i.e., decreases in macrophages for both the 0.05 µm and 1.0 µm particle sizes, and increases in neutrophils and the inflammatory cytokines IL-1 and MCP-1 for the 1.0 µm C60), the systemic immune response was unaffected by C60 exposure.