Document rBypmVRzK9eZmDzXZ4yegpqQE

M T D National Toxicology Program U.S. Department of Health and Human Services Filed in Fourth Judicial District Court 11/17/2017 6:55 PM Hennepin County, MN NTP M onograph Immunotoxicity Associated with Exposure to Perfluorooctanoic Acid or Perfluorooctane Sulfonate September 2016 / w\. 27-CV-10-28862 Systematic Review of Immunotoxicity Associated with Exposure to PFOA or PFOS Filed in Fourth Judicial District Court 11/17/2017 6:55 PM Hennepin County, MN hazard identification conclusion of p r e s u m e d t o b e a n i m m u n e h a z a r d t o h u m a n s or PFOS exposure is presumed to suppress the antibody response in humans. Human body of evidence: Moderate Confidence = Moderate Level of Evidence Animal body of evidence: High Confidence = High Level of Evidence Initial hazard conclusion (M oderate x High) = Presumed to be an Immune Hazard to Humans Final hazard conclusion (after consideration of biological plausibility) = Presumed to be an Immune Hazard to Humans Taken together, the human and animal bodies of evidence present a consistent pattern of findings that higher prenatal, childhood, or adult serum concentrations of PFOS are associated with suppression of the antibody response. Mechanistic data from in v i t r o or in v i v o studies can then be used to examine the biological plausibility of PFOS-associated suppression of the antibody response to develop the final hazard identification conclusion. The antibody response begins with B-cell surface antibody recognizing a specific antigen. Then, for a Tcell dependent antibody response (e.g., most of the experimental animal data on SRBC-specific antibody response), T-cells must also recognize the specific antigen involved (generally after processing by a macrophage, dendritic cell, or other antigen presenting cell). When B- and T-cells both recognize the same antigen, the T-cell activates the B-cell and releases cytokines to help the B-cell multiply and mature into an antibody secreting plasma cells that produces the antigen-specific antibody response. Therefore, relevant mechanistic data would include effects of PFOS at relevant concentrations on key cell populations (B-cells, T-cells, or macrophages as antigen presenting cells), antigen processing and cell activation, or cytokines important for cell signaling during the antibody response. PFOS-related decrease in B-cell or T-cell numbers would present a possible mechanism for reduced antibody response if it was observed at the same or lower concentrations at which reduced antibody response was observed. However, there is inconsistent evidence of reduced B-cell number (see B cells (B220)) at higher exposure levels fo r PFOS (>2 mg/kg) and no change in B-cell or T-cell (CD4 or CD8 subpopulations) numbers observed at lower doses PFOS (0.00166 to 0.08 mg/kg) associated with decreased antibody levels (see cell phenotyping data, spleen and thymus cellularity in Appendix 5 ) (Dong e t a l. 2009a, Zheng e t a l. 2009, Fair e t a l. 2011). Similarly, at lower exposure levels of PFOS, there are no changes in percentage or cell numbers of macrophages or other antigen presenting cells (Qazi e t a l. 2009a, Fair e t a l. 2011, Dong e t a l. 2012). Overall changes in leukocyte numbers and cellularity in the spleen and thymus are also not affected at lower doses of PFOS. There is no evidence that PFOS-induced changes in cell populations could explain the reduced antibody response at lower doses. Cytokine release of interleukin-4 (IL-4), IL-5, and IL-6 by T-cells are important fo r T-cell dependent antibody response (e.g., to SRBC). In studies designed to examine these cell signaling pathways in mice, PFOS exposure in v iv o resulted in increased IL-6 secretion from B-cells under culture conditions designed to test cytokine communication necessary for the antibody response (Fair e t a l. 2011) (i.e., including stimulation of the CD40 cell surface protein critical to IL-6 stimulation of IgM secretion) (Baccam e t a l. 2003, Bishop and Hostager 2003). Under these same conditions, PFOS did not affect IL-4, IL-5, or IL-6 secretion by T-cells in mice (Fair e t a l. 2011) (see cytokine data in Appendix 5 ). In a separate set of studies, PFOS exposure was associated with increased secretion of IL-4 and IL-6 in mixed cultures of splenocytes from mice exposed to higher doses (0.833 to 20 mg/kg/day), doses that are at or above PFOS doses associated w ith decreased antibody response (Dong e t a l. 2011, Mollenhauer e t a l. 2011, Zheng e t a l. 2011). Similarly, PFOS exposure was associated w ith increased IL-6 secretion in cultures of peritoneal macrophages from mice (Qazi e t a l. 2009a, Mollenhauer e t a l. 2011, Dong e t a l. 2012). As 64