Report on the Immunotoxicity Evaluations of Samples from Male and Female Harlan Sprague Dawley Rats Exposed to Specular Hematite by Inhalation for up to 39 Weeks
Report Date: September 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 The findings have not been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by NTP in March 2009.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 Toxicology Northwest (Battelle), Richland, WA, under Battelle Protocol No. 20536-02 as a part of National Toxicology Program Contract No. N01-ES-55534. At the request of the NTP, an immunotoxicological evaluation was conducted at Virginia Commonwealth University, Richmond, Virginia, under NTP Contract No. N01-ES-55538. The Study Director/Principal Investigator for the immunotoxicology studies was Kimber L. White, Jr., Ph.D., while Tai L. Guo, Ph.D., DABT, served as the Assistant Study Director.
The objective of the studies described in this report was to determine the potential effects of Specular Hematite (Chemical Abstracts Service #HEMATITESPEC) on the immune system of Harlan Sprague Dawley rats. Female HSD rats were exposed to specular hematite by inhalation at four concentrations (0, 15, 30, and 60 mg/m3) for up to 27 weeks. Male HSD rats were exposed to specular hematite by inhalation at four concentrations (0, 15, 30, and 60 mg/m3) for up to 39 weeks. The evaluation of immune parameters was conducted on samples collected from two groups: an Immunotoxicology Group (female HSD rats) and a Core Group (male HSD rats). The animals from the Immunotoxicology group were further divided into two subgroups. One subgroup was immunized with sheep erythrocytes and was used to evaluate the T-dependent antibody-forming cell response to sRBC and serum anti-sRBC IgM antibody levels, while the second subgroup remained unimmunized and was evaluated for immune cell subpopulations in the spleen, anti-CD3 mediated proliferation, natural killer cell activity, bronchoalveolar lavage fluid cytokine levels, and serum autoantibody levels (i.e., anti-nuclear antibodies. The Core Group was evaluated for BAL fluid cytokine levels and for serum autoantibody levels only. Samples from the Immunotoxicology Group were received from Battelle at pre-determined time points over the duration of the study (4 and 26 weeks for unimmunized animals; 5 and 27 weeks for immunized animals). Samples from the Core Group were received from Battelle at four pre-determined time points during the course of the study (4, 16, 26, and 39 weeks after study initiation).
On the day of study termination, spleens were placed in tubes containing media, placed on crushed ice, and shipped to VCU in Richmond, VA, for immunotoxicological evaluation the following day. The collected BAL fluid and the serum for autoimmunity evaluation were frozen and shipped separately on dry ice to VCU, where they were maintained at -70° C until evaluated. Mediastinal and tracheobronchial lymph nodes from unimmunized animals in the Immunotoxicology Group received in RNAlater from Battelle were stored at -70°C until requested for transfer to an NTP-designated facility for RNA evaluation. No significant 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 immunotoxicity of specular hematite.
No significant effects were observed in any of the immunological assays, including the AFC response, serum IgM antibody levels against sRBC, spleen cell numbers, spleen cell phenotypes, anti-CD3 mediated proliferation, or NK cell activity following exposure to specular hematite. The only exceptions were significant increases in the basal (unstimulated) proliferation of spleen cells at the 15 and 30 mg/m3 doses after 4 weeks of exposure and sporadic increases in NK cell activity after 26 weeks of exposure. BAL fluid cytokine levels were also unaffected, except for MCP-1, which was significantly increased in a dose-dependent manner after 26 weeks of exposure. Autoantibody levels were unaffected.
Core Group (male HSD rats): After 4, 16, 26, and 39 weeks of exposure to specular hematite, BAL fluid cytokines were not affected, with the exception of MCP-1, which was significantly increased in a dose-related manner at doses ≥ 30 mg/m3, beginning at the 16-week time point. ANA levels in the serum were unaffected.
In conclusion, specular hematite exposure by inhalation for up to 26 weeks in female HSD rats produced no systemic immunotoxic effects, as indicated by the absence of effects on the various components of humoral, cell-mediated, and innate immunity examined in these studies. MCP-1 was the only cytokine in the BAL fluid that was affected. Levels of MCP-1 were increased in a dose-dependent manner in both male and female HSD rats, beginning at week 16 in males and at week 26 in females. ANA levels in the serum were not affected by exposure to specular hematite.