An international validation study of the IL-2 Luc assay for evaluating the potential immunotoxic effects of chemicals on T cells and a proposal for reference data for immunotoxic chemicals

SUMMARY To evaluate the immunotoxic effects of xenobiotics, we have established the Multi-ImmunoTox assay, in which three stable reporter cell lines are used to evaluate the effects of chemicals on the IL-2, IFN-γ, IL-1β and IL-8 promoters. Here, we report the official validation study of the IL-2 luciferase assay (IL-2 Luc assay). In the Phase I study that evaluated five coded chemicals in three sets of experiments, the average within-laboratory reproducibility was 86.7%. In the Phase II study, 20 coded chemicals were evaluated at multiple laboratories. In the combined results of the Phase I and II studies, the between-laboratory reproducibility was 80.0%. These results suggested that the IL-2 Luc assay was reproducible both between and within laboratories. To determine the predictivity, we collected immunotoxicological information and constructed the reference data by classifying the chemical into immunotoxic compounds targeting T cells or others according to previously reported criteria. When compared with the reference data, the average predictivity of the Phase I and II studies was 75.0%, while that of additional 60 chemicals examined by the lead laboratory was 82.5%. Although the IL-2 Luc assay alone is not sufficient to predict immunotoxicity, it will be a useful tool when combined with other immune tests.

Pregnant Balb/C mice were treated with DPH at doses of 0, 20, 40, and 60 mg/kg via oral gavage on days 9 through 18 of gestation. A dose-related suppression of humoral immune function (measured as the antibody response to type III pneumococcal polysaccharide) was observed in male and female offspring at 25 days, but not at 15 weeks of age (NOAEL = 20 mg/kg). Female offspring of dams treated with 20 or 60 mg/kg DPH had greater antibody levels than controls. No difference was noted in female offspring of dams treated with 40 mg/kg DPH when compared to controls. Cell-mediated immune function (as measured by delayed-type hypersensitivity response to oxazolone) was not affected in offspring of treated dams. Immunosuppressive effects also were greater in offspring born with an open eye defect, also attributed to DPH treatment (Chapman and Roberts 1984).
Female B10.s, B10.d2 and DBA/2 mice were injected with 2 mg DPH and received a single injection of 10 µg TNP-OVA subcutaneously into the right hind footpad. Popliteal lymph nodes (PLN) were isolated 7 days after injection. DPH increased the number of cells in all three strains (B10.s>B10.d2>DBA/2) (data in graph). IgG1 production to TNP-OVA was increased in all three mouse strains (in B10.d2 about 850-fold; and in B10.s and DBA/2 about 120-fold). DPH treatment did not facilitate immune complex deposition in any of the mouse strains, six days after challenge (Albers et al. 1999).
DPH (administered subcutaneously) produced a significant, dose-dependent response in the PLN assay at 0.5 mg (mean PLN index =1.60 ± 0.18) and 1.0 mg (mean PLN index = 2.79 ± 0.30) as compared to control (mean PLN index = 1.11 ± 0.24) in C3Hf mice. The maximal response occurred at 6-8 days post treatment and returned to normal after 3-4 weeks. The observed response was proposed to be T-lymphocyte dependent since only heterozygous C3H +/nu mice developed PLN enlargement whereas their congenitally athymic C3H nu/nu counterparts did not.
In B6C3F1 mice exposed to acetonitrile by inhalation for 13 weeks, lymphoid depletion and lymphocytolysis in the thymus, spleen and bone marrow was reported in animals that died. A lack of immune effects were reported in mice exposed to acetonitrile for 2 years (NOAEL = 200 ppm) (National Toxicology Program 1996).
Based on a 14-day inhalation study in B6C3F1 mice (doses not provided), acetonitrile was not identified as an immunotoxicant (Luster et al. 1992).
Male Wistar rats were subcutaneously injected with acetonitrile at a dose of 0.8 LD50 (dose not provided). Antibody titer to sheep erythrocytes was decreased by 43%. Additionally, the number of antibody producing cells against sheep erythrocytes and Vi-Ag (no further information provided in article) were decreased by 52% and 27%, respectively. Thymus T-cell count, percentage of natural cytotoxicity (used as a surrogate for NK cell activity), and antibodydependent cell cytotoxicity also were significantly decreased after acetonitrile exposure. The percentage decreases were calculated as 31%, 52%, and 41%, respectively (Zabrodskii et al. 2002).

In vitro data with cells or cell lines
No data were located.
2 weeks for 6 weeks. B(a)P had no effect on the number of macrophages or lymphocytes in BAL from male or female offspring not treated with OVA. Additionally, no effect was noted on the number of macrophages or lymphocytes in B(a)P-treated offspring that were immunized with OVA (when compared with offspring only treated with OVA). IL-4, IL-5, IL-13, IL-33, and IFN-γ levels in the BAL were not affected in offspring not treated with OVA. Increased IL-33 and IFN-γ levels were observed in OVA-sensitized female offspring lactationally exposed to 5.0 and 100 pmol/week B(a)P, respectively. Lactational exposure to 0.25 B(a)P increased the total number of mediastinal lymph node cells in males. Lactational 100 pmol/week B(a)P increased numbers of TCRβ+ and CD86+ cells compared with vehicle in non-sensitized male offspring. In non-sensitized female offspring, lactational exposure to 100 pmol/week B(a)P increased numbers of CD11c+ PDCA-1-, CD28+, TCRβ+CD28+, MHC Class II+, and MHC Class II+CD86+ cells. In OVA-sensitized female offspring, a significant increase in CD11c+PDCA-1+ and CD11c+PDCA-1-cells was observed after exposure to 0.25 and 5.0 pmol/week, respectively (Yanagisawa et al. 2018).
Pregnant C3H/HeB mice were administered 150 mg/kg B(a)P via intraperitoneal injection on GD 11; immune effects were assessed at parturition and again one week after parturition. A significant reduction in newborn CD4+CD8+ (46%), CD4+CD8+Vγ2+ (60%), and CD4+CD8+Vβ2+ (53%) thymocytes were noted. Additionally, CD4+ splenocytes from 1-week-old offspring were significantly reduced (50%) (Rodriguez et al. 1999). B6C3F1 mice were administered 0.4, 4.0, or 40 mg/kg B(a)P by intratracheal instillation for seven days and immunized with sheep erythrocytes after the last B(a)P exposure. Decreased formation of antigen-specific AFC (by 60%) was observed at 40 mg/kg B(a)P in LALN. When sheep erythrocytes were administered by intraperitoneal injection, an increase in antigen-specific AFC was observed at 40 mg/kg B(a)P in LALN. However, the levels of AFC in the spleen were decreased (Schnizlein et al. 1987). B6C3F1 mice (3-6 months, 13-16 months, and 23-26 months) were administered 40 mg/kg B(a)P for 8 days by intraperitoneal injection. Mice also were immunized with sheep erythrocytes after day 4 of the B(a)P treatment. Spleens were removed and splenocytes assessed for formation of AFCs. Decreased formation of AFCs was noted in splenocytes from all three age groups. In two sets of experiments, the observed decreases were 23%-43% in mice ages 3-6 months, 63%-84% in mice ages 16-18 months, and 93% in mice ages 23-26 months (Lyte and Bick 1985). B6C3F1 mice were administered 5, 20, or 40 mg/kg B(a)P for 14 days by subcutaneous injection. Spleens were removed and ConA-induced production of IL-2 and IL-3 were assessed. While splenocyte IL-2 production was decreased in a dose dependent manner, no effect on splenocyte IL-3 production was noted. As shown in other studies, B(a)P decreased responses to sheep erythrocytes (>95% inhibition). Addition of exogenous IL-2 to the treated splenocytes, reversed the B(a)P-induced inhibition of responses to sheep erythrocytes (Lyte et al. 1987;Lyte and Bick 1986).
Female B6C3F1 mice were administered 10 subcutaneous injections of B(a)P over a 14-day period at doses of 5, 20, or 40 µg/g. KLH-sensitization did not affect delayed hypersensitivity responses at the tested doses. Additionally, B(a)P treatment did not induce rejection to DBA mice skin that was grafted onto mice. Proliferative responses to PHA were dose-dependently decreased (LOAEL = 20 µg/g B(a)P). Spontaneous and LPS-induced proliferative responses were increased at 5 µg/g B(a)P and significantly decreased at 40 µg/g B(a)P. MLC responses, and the percentage of spleen cells with T-and B-cell surface markers were not significantly affected at any of the tested doses. Additionally, NK cell activity against YAC-I target cells was not impacted in mice treated with 40 µg/g B(a)P (data not provided). Serum IgG levels were dose-dependently decreased in treated mice (18-24%). A reduction in the number of antibody plaque forming cells to sheep erythrocytes and LPS were noted (LOAELs = 20 and 5 µg/g B(a)P, respectively). B(a)P exposure decreased response to TNP-Ficoll without effects on TNP-LPS response. Host resistance studies showed that B(a)P had no effect on PYB6 tumor incidence or susceptibility to L. monocytogenes (Dean et al. 1983).

In vitro data with cells or cell lines
Rat and mouse spleen cells were treated with B(a)P for 24 hours and then LPS or PHA for 48 hours. Then, mitogen stimulation was assessed by 5-bromo-2'-deoxyuridine incorporation. B(a)P inhibited cellular proliferation for both species at similar concentrations (data in graphs). B(a)P also inhibited rat spleen proliferation that was stimulated by ConA (data provided in graph). B(a)P inhibited anti-CD3 antibody stimulation of mouse lymphocyte proliferation (IC50 > 160 µM) ). B(a)P decreased viability of mouse antigen presenting cells (APC) and increased expression of CD86 expression on APC (LOAELs = 0.1 µM). In murine splenocytes, B(a)P decreased cell viability and proliferation (LOAELs = 0.1 and 1.0 µM, respectively). B(a)P did not modulate the expression of T-cell receptors or CD19 at any of the tested concentrations in murine splenocytes (Chowdhury et al. 2017). B(a)P decreased ConA induced cellular proliferation of mouse splenic T-cells in a dosedependent manner (LOAEL = 0.1 µg/mL). Inhibition of IL-2, IL-4, and IFN-γ also was observed in ConA-stimulated splenic T-cells (LOAELs = 0.1, 0.2, and 0.1 µg/mL, respectively) (Guan et al. 2017). B(a)P inhibited spleen cell response to sheep erythrocytes in a concentration dependent manner (LOAEL = 0.01 µM). B(a)P also inhibited one-way mixed lymphocyte response with a maximal inhibition of 19% (Urso et al. 1986). Similar response of murine spleen cell response to sheep erythrocytes was reported by Kawabata and White (1987) (LOAEL = 1 nM) after incubation for 5 days.
Splenocytes from B6C3F1 mice (3-6 months and 23-26 months) were exposed to 1, 10, or 50 µg/mL B(a)P and sheep erythrocytes for 4-5 days. After end of exposure period, the number of AFCs was determined. Dose-dependent decrease in the number of cells was observed in splenocytes from both age groups (data in graphs) (Lyte and Bick 1985). B(a)P (in PVP-NaCl) dose-dependently increased LPS-induced IL-1 production by peritoneal exudate macrophages isolated from B6C3F1 mice; tested concentrations ranged from 25 to 800 µg/mL. A concurrent decrease in cell viabilities was noted at the same test concentrations. Comparatively, when B(a)P was dissolved in corn oil no effect on IL-1 production or cell viabilities was noted (Lyte and Bick 1986).

Mode of action information
Disruption of T-cell activities has been associated with B(a)P induced immunotoxic effects (Urso et al. 1986). Modulation of mouse splenic T-cell effects was associated with modulation of calcium levels; which was associated with suppression of the NF-B and NFAT pathways (Guan et al. 2017).
In addition to T-cell effects, modulation of B-cell population or responses, or macrophage functions also have been implicated in B(a)P mode of action (Saxena et al. 2018;Urso et al. 1986). Hardin and colleagues (1992) proposed that B(a)P-induced suppression of B-cell lymphopoiesis was, partially, produced through induction of programmed cell death. Ah-receptor dependent-and/or independent-pathways could produce the observed effects.

Human Data
Data from epidemiology studies No data were located.

In vitro data with cells or cell lines
Cadmium chloride (10-100 µM) inhibited NK (against K562 cells) and antibody-dependent cellular (against P815 cells) cytotoxicity (ADCC) in peripheral blood lymphocytes in a concentration-dependent manner. The estimated 50% inhibition doses (ID50) for NK and ADCC activities were 50 and 100 µM, respectively. NK and ADCC activities were not significantly affected by changing the effector cell:target cell ratios. Cadmium chloride also inhibited cytotoxic activity against K562 or Daudi cells in activated IL-2 cells (data in graph). Time-course studies showed that a significant decrease in NK and ADCC activities was observed when added at 90 minutes after the start of the experiment (Cifone et al. 1990).

Mode of action information
In vitro studies suggest that in peripheral blood lymphocytes, cadmium chloride modulated phosphoinositide hydrolysis induced by a target molecule. This modulation is proposed to lead to inhibited NK activity (Cifone et al. 1990).
Proposed direct action of cadmium on immunocompetent cells stimulates production and release of cytokines, which may produce proinflammatory effects (Marth et al. 2000).

Rodent Data
Data from in vivo immunotoxicology or toxicology studies Female BDF1 mice were provided drinking water containing 5, 10, or 50 µg/mL cadmium (as cadmium chloride) for 3 weeks. Antibody response to sheep erythrocytes was decreased in a dose-dependent manner. Splenic plaque-forming cell number decreased by 16% to 28%. A dosedependent increase in LPS-induced proliferation also was observed (LOAEL = 10 µg/mL). In the absence of a mitogen, cadmium chloride also increased lymphocyte proliferation (LOAEL = 10 µg/mL). No effect was observed when ConA mitogen was used to stimulate proliferation (Blakley 1985).
Female CD1 mice were provided drinking water containing 5, 10, or 50 µg/mL cadmium (as cadmium chloride) for 3 weeks. In vivo T-lymphocyte independent (against DNP-Ficoll) and T-lymphocyte and macrophage independent (against E. coli) responses were increased by cadmium exposure (Blakley and Tomar 1986).
Female BDF1 mice were provided drinking water containing 50 µg/mL cadmium (as cadmium chloride) for 3 weeks. Spleen cell suspensions from pooled spleens were separated by adherence techniques and antibody production against sheep erythrocytes was assessed. Suppressed antibody production (26%-34%) was noted in cultures that contained cadmium-exposed Tlymphocytes. Antibody production was similar to controls in cultures that contained cadmiumexposed macrophages (Blakley and Tomar 1986).
Male Sprague-Dawley rats were administered 0.7 or 6 mg/kg cadmium (as cadmium chloride) by oral gavage for 28 days. Splenocyte proliferation was significantly decreased (76% of control) in rats that were administered 6 mg/kg cadmium. Splenocyte IL-2 production also was increased after administration of 6 mg/kg cadmium, when production was normalized with cell number. No effect was noted on splenocyte IFN-γ production (Wang et al. 2017).
Immunotoxic effects in offspring were noted after exposure to cadmium chloride in utero or through milk. In ICR mice administered 2.5 or 5.0 mg/kg cadmium chloride on GD 16, a significant increase in offspring spleen weight was reported (LOAEL = 2.5 mg/kg). Unstimulated spleen lymphocyte proliferation was significantly increased at both tested doses (1.5-to 2-fold). Additionally, ConA, PHA, and LPS stimulation was increased in treated animals (LOAELs = 5.0, 5.0, and 2.5 mg/kg). No effect on delayed-type hypersensitivity to sheep erythrocytes was reported, but an increase in total Ig and IgM antibody titer was noted at 2.5 mg/kg (Soukupova et al. 1991). In offspring that were exposed to cadmium chloride through maternal milk (dams received 5 ppm or 10 ppb cadmium chloride in water until weaning) decreased spleen weights were observed in females, but not males (data in graphs). The effect was greater in lower dosed females. Effects on organ weight did not persist to adulthood. In adult and juvenile rats, effects on cytotoxic activity of splenic NK-cells was noted (data in graphs). Additionally, cadmium chloride inhibited ConA-induced thymocyte proliferation in both male and female adult rats (Pillet et al. 2005).
Female C57BL/6 mice were exposed (nose-only) to aerosolized cadmium chloride (60-minute exposure to 0.88 mg Cd/m 3 ) and examined 5-18 days later. Decreased splenic cell viability was observed (data in graph). Significant decreases of proliferative responses to LPS and PHA, and inhibition of IgM secretion in response to sheep erythrocytes were observed. Comparatively, oral chronic exposure (5, 100, or 300 ppm cadmium chloride in water for 12-16 weeks) suppressed IgM response to sheep erythrocytes, without effects on cell viability (Krzystyniak et al. 1987).

In vitro data with cells or cell lines
Splenocytes isolated from male Sprague-Dawley rats were treated with ConA for 24 hours, followed by incubation with 5, 10, or 20 µM cadmium chloride for 4 or 24 hours. After exposure for 4 hours, decreased IL-2 (LOAEL = 5 µM) and IFN-γ (LOAEL = 10 µM) production was observed in the absence of effects on cell proliferation. After exposure for 24 hours, decreased IL-2 (LOAEL = 5 µM) production and cell proliferation (LOAEL = 10 µM) were observed. When cytokine production after 24-hour exposure was normalized based on cell number, August 8, 2018 increased IFN-γ production (LOAEL = 10 µM) was noted. For IL-2 production, a significant decrease was noted at 5 µM and an increase was noted at 20 µM (Wang et al. 2017).

Mode of action information
In RAW264.7 cells, cadmium chloride upregulation of COX-2 and MIP-2 was associated with activation of the phosphatidylinositol 3-kinase/Akt signaling pathway (Huang et al. 2014).
Cadmium chloride also may induce overstimulation of nuclear factors of activated T-cells to activate Jurkat T cells (Colombo et al. 2004).

In vitro data with cells or cell lines
In cultured PBMCs collected from healthy, non-smoking volunteers and cultured in DBAA for four hours, DBAA increased the percentage of necrotic human PBMC and decreased PBMC cell size (LOAEL = 5 mM). Increases in the percentage of apoptotic cells and PBMC granulation also was reported (LOAEL = 1 and 5mM, respectively). Caspase-8, -9, and -3 expression were upregulated at 1 and 5 mM. Increased transmembrane mitochondrial potential and levels of reactive oxygen species (ROS) also were noted with DBAA exposure (LOAEL = 1 and 0.1 mM) (Michalowicz et al. 2015).

Mode of action information
DBAA may increase ROS levels and transmembrane mitochondrial potentials (Michalowicz et al. 2015).

Data from in vivo immunotoxicology or toxicology studies
In female F344/N rats exposed to DBAA for 3 months (0-2000 mg/L in drinking water), minimal to mild hematopoietic cell proliferation was noted at the highest dose. A similar effect was not observed in males. While no spleen effects were noted in B6C3F1 mice exposed to DBAA for 3 months (0-2000 mg/L in drinking water), thymus atrophy was reported in males and females (LOAEL = 1000 and 2000 mg/L, respectively) (National Toxicology Program 2007).
In male and female BALB/c mice orally gavaged with 5, 20, or 50 mg/kg DBAA for 28 days altered spleen and thymus weights, and splenic and thymic cellularity were reported. DBAA also inhibited B-cell proliferation (LOAEL = 20 mg/kg). DBAA increased T-cell mitogenesis (value not provided) at 20 mg/kg. DBAA increased apoptosis in spleen and thymus in a dose-dependent manner (values not provided). Additionally, DBAA exposure altered the expression of apoptosisrelated genes in spleens and thymus of treated mice. In the thymus, expression of Fas and TRAF2 were altered (2-2.5 fold). In spleens of treated mice, expression of Fas and TRAF2 were increased 5-fold while bcl-2 expression was decreased 1.5-fold. Increased protein expression of Fas and FasL also were observed in spleen and thymus of treated mice (LOAEL = 5 mg/kg) (Gao et al. 2008). 7.97 ± 0.53 7.08 ± 0.74 5.42 ± 0.79* 4.28 ± 0.39*** Data are presented as mean ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05, significance assessed by ANOVA when compared with control group (DBA 0 mg/kg).
Increased neuronal expression of immune factors was noted in Sprague-Dawley rats administered 20, 50, or 125 mg/kg DBAA via intragastric injection for 4-weeks. mRNA expression of Iba-1, NK-B, IL-6, IL-1β, and TNF-α were increased in the pre-frontal cortex and hippocampus of treated rats (LOAEL = 50 mg/kg for all brain regions). Protein levels of Iba-1, NK-B, IL-6, IL-1β, and TNF-α also were significantly increased in the same brain regions. Protein expression LOAEL in the pre-frontal cortex for Iba-1, NK-B, IL-6, IL-1β, and TNF-α was 50 mg/kg. Protein expression LOAEL In the hippocampus was 100 mg/kg for NK-B and 50 mg/kg for other evaluated cytokines (Jiang et al. 2017).
Female B6C3F1 mice were given drinking water with 125, 500, or 1000 mg/L for 28 days. A significant decrease in thymus weight was noted at 500 and 1000 mg/L (19%). No effect on absolute or relative spleen weight, or relative thymus weight was reported. A non-dose response decrease (19%) in total spleen cell number and number of CD+CD-T-lymphocytes (13%) was observed at 500 and 125 mg/L, respectively. A significant decrease in absolute (38%) and percent (22%) NK1.1+CD3-cells was noted at 500 mg/L. Significant decreases in absolute and percent splenic macrophages also were observed (LOAEL = 500 and 1000 mg/L, respectively). No effects on absolute or percent Ig+, CD3+, CD4-CD+, or CD4+CD8+ markers were noted. No effects on AFC response or IgM antibody titers in response to exposure to sheep red blood cells were noted. Additionally, no impact on response to allogeneic spleen cell stimulation was noted. A significant decrease in cytotoxicity was only observed after splenocyte NK cell activity was augmented with poly-IC; the effect was only observed at 125 mg/L. Host resistance to Streptococcus pneumoniae, Plasmodoium yoelii, and B16F10 melanoma tumors was not affect by treatment (Smith et al. 2010).

In vitro data with cells or cell lines
DBAA decreased thymocyte (obtained from BALB/c mice) proliferation at exposure lengths of at least 6 hours. At 6 hours, a significant decrease in proliferation was only observed at 40 µM. Comparatively, at 12, 24, and 48 hour exposure periods a significant decrease in proliferation was observed at 5, 10, 20 and 40 µM. DBAA also decreased IL-2 and IL-4 secretion (LOAEL = 10 and 5 µM, respectively). DBAA also increased late and early apoptosis (LOAEL = 5 and 10 µM), without effects on the percentage of necrotic cells. DBAA induced an increase in the percentage of cells in the G0/G1 phase and decreased the percentage of cells in the S phase. Increased intracellular thymocyte calcium levels (LOAEL = 5µM) and thymocyte Fas and FasL protein levels were reported (LOAELs = 10 µM for both proteins). Additionally, bcl-2 protein level was significantly decreased at all tested concentrations (LOAEL = 5 µM) (Gao et al. 2016).
Peritoneal exudate cells, obtained from B6C3F1 mice treated with 125, 500, or 1000 mg/L DBAA for 28 days, were evaluated for their ability to suppress B16F10 melanoma tumor cell proliferation in vitro. Treatment did not affect the ability of macrophages obtained from treated animals to suppress proliferation (Smith et al. 2010).

Mode of action information
Overall, studies suggest that DBAA produces immunotoxic effects through modulation of T-cell mediated cell immunity. T-cell apoptosis, through extrinsic and intrinsic pathways, are proposed to play a role in the mode of action. Apoptosis may occur through a variety of pathways including modulation of transmembrane potential, the Fas/FasL pathway, modulation of intracellular calcium, and cell cycle arrest (Gao et al. 2008;Gao et al. 2016).

References
Gao S, Wang Y, Zhang P, Dong Y, Li B. 2008. Subacute oral exposure to dibromoacetic acid induced immunotoxicity and apoptosis in the spleen and thymus of the mice. In vitro data with cells or cell lines DBP significantly decreased phagocytotic capacity of differentiated THP-1 cells at all tested concentrations (LOAEL = 0.001µM). DBP also increased TNF-α secretion (LOAEL = 0.1 µM). Comparatively, DBP had no effect on IL-1β or IL-8 secretion from differentiated THP-1 cells (NOAEL = 0.001 µM) (Couleau et al. 2015).
High-density microarray studies were conducted using normal human mammary epithelial cell strains obtained from discarded tissues; cells were treated with 1 µM DBP for 10 hours. Gene expression of 29 genes were increased in all four isolated cell strains. Gene expression of 28 genes were decreased in all four isolated cell strains including genes involved in the immune response (TNF-α-induced protein 3; values not provided) (Gwinn et al. 2007).
DBP (tested at 0.1 and 100 µM) increased IL-6, CXCL8, and IL-10 secretion from monocytes/macrophages, isolated from blood of healthy individuals. The cells were, stimulated with E. coli lipopolysaccharide (LPS) for 1 hour. Comparatively, DBP did not affect IL-1β and decreased TNF-α secretion from the cells. For all affected cytokines the LOAEL was 100 µM. For phytohemagglutinin-P (PHA-P) stimulated T cells, DBP decreased IL-2, IL-4, TNF-α and IFN-γ secretion (LOAEL for all cytokines = 100 µM). No effect on IL-6 or IL-10 secretion was observed in the PHA-P stimulated T cells treated with DBP. Metabolism studies indicated that DBP was metabolized to monobutyl phthalate in vitro. Additionally, secretion patterns of monobutyl phthalate was similar to those observed for DBP (Hansen et al. 2015).
DBP increased IL-1β gene expression (as assessed by RT-PCR) in human corneal endothelial cell line B4G12 at all tested concentrations (LOAEL = 1 µM). IL-8 gene expression was increased at 1 and 10 µM (values not provided). IL-β, IL-8, and IL-6 secretion from cells also was increased. IL-6 and IL-8 LOAEL values were 10 and 5 µM, respectively. Significant IL-1β secretion was only observed at 1 µM. [Note: The authors note that secretion for IL-1β and IL-6 was low and quantification was approximate] (Kruger et al. 2012).

Mode of action information
Studies suggest that innate and adaptive immune system is impacted by DBP exposure (Hansen et al. 2015). DBP is proposed to be metabolized to the monoester in vitro. This compound then is proposed to modulate cytokine secretion from both monocytes/macrophages and T cells.
The results from Couleau and colleagues (2015) suggest that some effects may occur through activation of the endocrine pathway. DBP also may regulate gene and protein expression of a variety of immune factors (e.g., cytokines) without impacting cell viability.
Immunomodulation by DBP also may occur through receptor-mediated effects on the inflammasome.

Rodent Data
Data from in vivo immunotoxicology or toxicology studies DBP did not increase proliferative responses in lymph nodes of BALB/c mice at concentrations up to 20% (v/v in acetone; dermal route of exposure). Additionally, 10% DBP did not increase dendritic cell accumulation in draining lymph nodes (Dearman et al. 1996).
Wistar rats were fed a diet containing 0.5% or 5% DBP for 34-36 days. While no effect on absolute spleen weight was reported, a significant increase (1.8-fold) in relative spleen weight was reported at 5% DBP (Murakami et al. 1986).
Female BALB/cJ mice were subcutaneously exposed to ovalbumin (antigen) and 2-2000 µg/mL DBP. After the primary immunization, one or two booster shots were given to the mice. No effects on the IgG1 or IgE serum levels after either one or two booster shots were noted. A dosedependent effect was observed on IgG1 serum levels; maximum responses were observed at 200 µg/ml (value not provided). No effect was noted on IgE serum levels (data not provided) (Larsen et al. 2002).
Thymic stromal lymphopoietin (TSLP) mRNA expression was significantly increased in BALB/c mouse ears 24 hours after exposure to DBP (in acetone, 1:1) (values not provided). An increase in TSLP protein levels was also measured at 24 hours (values not provided) (Larson et al. 2010;Schuepbach-Mallepell et al. 2013). DBP-induced induction of TSLP was strain dependent (BALB/c was more sensitive than C57Bl/6 mice). DBP also produced effects on TSLP in IL-1 receptor or apoptosis-associated speck-like protein containing a caspase recruitment domain deficient mice (Schuepbach-Mallepell et al. 2013). August 8, 2018 In vitro data with cells or cell lines DBP was cytotoxic to murine peritoneal exudate macrophages (PEM) after exposure to 50 or 100 µM for 24 hours. Annexin V and PI double stained cells (markers of apoptosis) were significantly increased after treatment with 100 µM DBP for 24 hours. Additionally, using trypan blue exclusion, a significant decrease in viable cells was reported after DBP exposure (LOAEL = 50 µM). Using two-color flow cytometry, DBP was shown to decrease expression of CD80, CD36, and major histocompatibility-II molecules on F4/80+ macrophages at 1 and 10 µM. Cytokine expression (IL-1β, IL-6, IL-12, and TNF-α) also were decreased at the same concentrations. Phagocytotic capacity of PEM to apoptotic thymocytes and E. coli was decreased after exposure to DBP when compared to controls (LOAEL = 1 µM). DBP exposure also decreased PEM immunogenicity to allogenic T cells (LOAEL = 1 µM) (Li et al. 2013).
In RBL-2H3 mast cells sensitized with anti-dinitrophenyl monoclonal IgE, DBP potentiated βhexosaminidase activity, which was used as a measurement of degranulation (LOAEL = 50 µM for 10 minutes). DBP did not induce degranulation in the cells that were not sensitized (NOAEL = 500 µM for 10 minutes) (Nakamura et al. 2002).
In PAM212 keratinocytes, 1% DBP increased relative expression of TSLP; maximal effect (values not provided) was observed at 36 hours post treatment (Larson et al. 2010). DBP-induced TSLP expression was associated with epidermal mouse skin and human abdominal skin transplanted on mice (Schuepbach-Mallepell et al. 2013).

Mode of action information
In vivo rodent studies suggest that DBP impacts the Th2 response. Inflammasome activation by DBP impacts TSLP expression and Th2 response.
In vitro studies suggest that while high doses of DBP induced macrophage apoptosis, moderate doses induced protein expression and production of cytokines. DBP also impacted the antigenpresenting capacity of macrophages.

Data from epidemiology studies
No studies were identified.

In vitro data with cells or cell lines
A single study suggested that DCAA may produce immunosuppressive effects. Using a two-way mixed lymphocyte reaction, DCAA (LOAEL = 0.33 mM; lowest dose tested) increased IL-10 production and FOX P3 expression 11.4-and 4.5-fold, respectively (Eleftheriadis et al. 2013). DCAA (3.0 mM and 0.5 mM, respectively) increased IL-2 production after incubation for 16 hours and expression of the T-cell activation marker CD25 in Jurkat cells. Comparatively, no effect on CD69 expression (0.5 and 3.0 mM) was noted. IL-2 and IFN-γ mRNA expression was significantly increased after DCAA treatment (3.0 and 0.5 mM, respectively) (Pan et al. 2015).
DCAA (N/LOAEL = 0.1/1.0 mM) induced statistically significant increases in necrosis in PBMC, as shown by a decrease in PBMC cell size combined with an increase in cellular granulation. Statistically significant increases in the percentage of apoptotic cells were observed at similar concentrations of DCAA (N/LOAEL = 1.0/2.0 mM) (Michalowicz et al. 2015).

Mode of action information
T-cell activation was one proposed mode of action for DCAA. Increased IL-10 production, combined with increased FOX P3 expression, is proposed to increase regulatory T-cell differentiation which may lead to increased IL-10 production. Additionally, DCAA increased expression of T-cell activation markers in Jurkat cells.
Apoptosis was proposed be associated with a variety of mechanisms including ROS generation, alterations in mitochondrial transmembrane potential, and activation of caspase activity.

Data from in vivo immunotoxicology or toxicology studies
In a 90-day drinking water study (0, 50, 500 and 5000 ppm (w/v) DCAA) with male Sprague-Dawley rats, a significant increase in relative spleen weight was noted at 5000 ppm (0.25% vs. 0.21%). No consistent effects on T cell-dependent anti-keyhole limpet hemocyanin IgG antibody production (measured by ELISA), delayed hypersensitivity to bovine serum albumin, NK cell cytotoxicity, or production of peritoneal macrophage-derived PGE2 or spleen lymphocytederived IL-2 were detected at tested doses (data not shown in paper) (Exon et al. 1986;Mather et al. 1990).

In vitro data with cells or cell lines
No studies were identified.

Mode of action information
DCAA-induced increase of p53 accumulation has been proposed to lead to increased formation of cells in G2-M phase (Staneviciute et al. 2016).

Data from epidemiology studies
Male and female offspring of pregnant women given DES doses from the seventh week to 34 th week of pregnancy, were interviewed about immune-related health problems. A total of 549 DES-exposed offspring and 487 placebo-exposed offspring participated in the study. Rates of allergy-related health problems (e.g., asthma, drug allergy, hives) were similar between DESand placebo-offspring. Infection (e.g., shingles, flu) and autoimmune disease (e.g., diabetes, rheumatoid arthritis) also were similar between the two groups (Baird et al. 1996).
The frequency of any autoimmune disease in women exposed to DES in utero (n = 1711) was higher than the frequency observed in control women. The overall frequency was 28.6 per 1000 women compared to 16.3 per 1000 women. Hashimoto's thyroiditis was significantly more prevalent (relative prevalence = 5.4) in exposed women compared to controls (Noller et al. 1988).
Increased incidence of asthma, arthritis, and diabetes mellitus was reported in sons and daughters exposed to DES in utero when compared to unexposed individuals. Additionally, the number of respiratory tract conditions (e.g., colds) was increased in the exposed population vs. the unexposed population (Wingard and Turiel 1988).

In vitro data with cells or cell lines
Lymphocyte NK activity (assessed using chromium release from K562 cells) from 12 patients exposed to DES in utero was greater than observed from controls; however, effects were not significant. No effects on adherent cells were noted (Ford et al. 1983). Comparatively, DES dose-dependently inhibited lysis of K562 cells in PBMCs obtained from 12 patients. At the highest concentration tested (100 µM), an 82% reduction in activity was observed compared to control samples (Ablin et al. 1988b(Ablin et al. , 1988a Responses to 0.125 µg/mL PHA (as measured by uptake of radiolabeled thymidine) was significantly greater in peripheral blood monocytes from women exposed to DES in utero compared to controls (88.6 × 10 3 vs. 44.0 × 10 3 cpm; p < 0.002). Maximal blastogenic response to PHA in lymphocytes from DES-exposed women was observed at 0.125 µg/mL while it was observed at 0.25-0.50 µg/mL in controls (Ways et al. 1987).
Male and female CD-1 mice were subcutaneously injected with 5, 15, or 30 µg/kg DES, four times on alternate days. Relative thymic weight (LOAEL = 30 µg/kg) was decreased, and absolute and relative splenic weights (LOAELs = 15 µg/kg) were increased in female mice. A similar effect in male mice was not observed. Relative expression of thymocyte populations (e.g., CD4-8-) were not affected in males or females. However, an increase in the number of total apoptotic and decrease in the number of live CD4+8+ and CD4+8-thymocytes was observed in male and female mice. Additionally, an increase in the number of total apoptotic and decrease in the number of live CD4-8-cells were observed in females. Increased proliferative response to ConA, LPS, or PMA was observed in splenic lymphocytes isolated from female mice treated with 5 µg/kg DES. At higher doses, a trend for decreased proliferation was observed in female splenic lymphocytes. Proliferative responses by splenic lymphocytes were only modulated in response to ConA at 15 µg/kg DES (Calemine et al. 2002).
Female mice (strain not provided) were administered (route not provided) 0.2, 2.0, or 8.0 mg/kg DES for 5 days. Antibody response to sheep erythrocytes and LPS were decreased 15% to 45% (LOAELs = 2.0 mg/kg). Delayed hypersensitivity response to keyhole lymphet hemocyanin was similar to controls when mice were exposed to DES before sensitization. However, when mice were exposed to DES after sensitization and before challenge a decrease in response was observed (LOAEL = 2 mg/kg). The percentage of splenic T lymphocytes was decreased 25% at the highest dose tested. No effect on the percentage of splenic B lymphocytes was observed. Splenic lymphoproliferative response to PHA and ConA were decreased (>30%) at all tested doses. Responses to Staphylococcus enterotoxin A were increased at 0.2 mg/kg and decreased at higher doses, while responses to LPS were increased at 0.2 and 2.0 mg/kg and decreased at 8.0 mg/kg. MLC responses also were decreased (LOAEL = 2 mg/kg). Suppressor cell activity was decreased after exposure to 8 mg/kg DES (Luster et al. 1980). Differential effects on the immune system were observed in female NMRI mice depending on the time of DES exposure. Thymus weights were increased in 56-day-old mice that were subcutaneously injected with 5 µg from PND 1-5, 6-10, or 30-34 (1.2-to 1.4-fold). Comparatively, thymus weight was decreased in mice subcutaneously injected with DES from PND 48-52 (29%). A dose-related effect on thymus weight was observed in mice treated from PND 1-5; no effects on absolute or relative spleen weight were noted. Differences in thymus weight also were noted depending on when the mice were killed after treatment. Four days after treatment, thymus weights were decreased in all test groups. However, 4 to 8 weeks after treatment showed an increase in thymus weight in mice treated on PND 1-5 and weights similar to controls in other treatment groups. DES treatment on PND 1-5 also reduced the number of cells in S-phase in the thymus (Forsberg 1996). C57BL/6 mice were treated with DES once in utero and/or once at 12-16 months of age via subcutaneous injection. Increased secretion of IFNγ was observed in splenic lymphocytes obtained from mice exposed to DES in utero and as adults. Increased IFNγ also was observed when splenocytes were stimulated with anti-CD3 antibodies. This increase was not observed in other treatment conditions (data in graphs). An increase in IFNγ production also was observed in T-cells from mice exposed to DES in utero and as adults (Karpuzoglu-Sahin et al. 2001).
DES increased IgE levels in male BALB/c mouse splenocytes at concentrations greater than 1 µM. Comparatively, DES had no effect on IgM, IgG, or IgA levels at concentrations up to 1 mM (Han et al. 2002).

Mode of action information
DES-induced thymic atrophy was proposed to be due, in part, to estrogen-related thymocyte apoptosis (Besteman et al. 2005;Fenaux et al. 2004). Brown and colleagues suggested that DES exposure upregulates TNF family members, which leads to altered T-cell development. This alteration was suggested to lead to thymic atrophy (Brown et al. 2006). Direct effects on T lymphocytes also may occur (Luster et al. 1980).
In mice, DES exposure was associated with down-regulation of gene expression in the TCR complex, and the TCR and CD28 signaling pathways. Genes in the B-cell receptor signaling pathway, antigen presentation, and dendritic cell pathways also was altered by DES exposure. It was proposed that DES dysregulation of T-cell development plays a role in thymus effects (Frawley et al. 2011). Alterations of microRNA expression also has been proposed as playing a role in the immunotoxic effects produced by DES (Singh et al. 2015).
Additional proposed modes of action on the immune system include effects on adherent suppressor cells, modulation of NK activity by interfering with bone marrow lymphoid precursors, and modulation of the mononuclear phagocyte system (Dean et al. 1986;Forsberg 1984;Kalland 1984;Luster et al. 1980).

Human Data
Data from epidemiology studies The prevalence of adult-onset asthma, in relation to lifetime pesticide use, were assessed using data from the Agricultural Health Study (19,704 male farmers). Adult-onset asthma was reported in 441 individuals; 127 classified as allergic and 314 classified as non-allergic. EDB exposure was positively associated with allergic asthma (OR: 2.07 [1.02-4.20]) (Hoppin et al. 2009).

In vitro data with cells or cell lines
No data were located.

Mode of action information
No data were located.

Data from in vivo immunotoxicology or toxicology studies
Female B6C3F1 mice were intragastrically treated with 100, 125, 160, or 200 mg/kg EDB for 14 days. Relative thymus and spleen weights were decreased in a dose-related manner (LOAEL = 200 mg/kg). Comparatively, relative liver and kidney weights were increased at higher doses (LOAEL = 125 and 160 mg/kg, respectively). Significant increases in white blood cells (LOAEL = 200 mg/kg) and neutrophils (LOAEL = 160 mg/kg) were noted. Host resistance to influenza A2, Listeria monocytogenes, and herpes simplex virus types 1 and 2 was not significantly affected by EDB exposure. The total number of resident peritoneal exudate cells were significantly increased in EDB-treated mice (LOAEL = 160 mg/kg). However, the percentage of cell types present in the exudates were similar to those observed in control exudates (macrophages: 53%; lymphocytes: 47%). Phagocytosis of radiolabeled chicken red blood cells was increased in peritoneal macrophages obtained from EDB-treated mice (187% of control; LOAEL = 125 mg/kg). Splenic NK cell activity was evaluated in animals treated with 100, 125, or 160 mg/kg; a significant decrease in activity was observed at 160 mg/kg. The number of viable cells in the spleen decreased at 125, 160, and 200 mg/kg (not significant), while a significant increase in the number of anti-SE PFC/10 6 viable spleen cells was significantly increased at 160 mg/kg. Splenic lymphocyte responses to allogenic spleen cells, PHA and ConA, but not LPS, were significantly decreased at 125 and 160 mg/kg (Ratajczak et al. 1994).
Female B6C3F1 were intragastrically treated with 31.25, 62.5, or 125 mg/kg EDB for 5 days per week for 12 weeks. No effect on white blood cell numbers, or the percentage of neutrophils or lymphocytes were noted at the doses tested. Splenic lymphocyte responses to PHA and LPS were significantly decreased at the highest dose tested (data not provided) (Ratajczak et al. 1995).
Relative spleen weights were not significantly affected in male Sprague-Dawley rats inhalationally exposed to EDB 7 hours per day, 5 days per week, for 30 days. However, relative liver weights were increased at the highest dose tested (LOAEL = 455 ppm) (Igwe et al. 1986).

In vitro data with cells or cell lines
No data were located.

Mode of action information
No data were located.

Human Data
Data from epidemiology studies No data were located.

In vitro data with cells or cell lines
No data were located.

Mode of action information
No data were located.

Data from in vivo immunotoxicology or toxicology studies
Of 10 F344/N female rats that received 400 mg/kg glycidol for 13-weeks (via gavage), lymphoid necrosis of the thymus was observed in nine (Irwin et al. 1996;National Toxicology Program 1990). Enlarged spleen were observed in haploinsufficient p16 Ink4a /p19 Arf male mice treated with 200 mg/kg glycidol for 40 weeks via gavage (National Toxicology Program 2007).
Increased splenic fibrosis incidence was reported in male and female F344/N rats gavaged with 37.5 and 75 mg/kg glycidol for 2 years. In males, splenic fibrosis incidences were 26% in controls, 68% in rats treated with 37.5 mg/kg, and 56% in rats treated with 75 mg/kg. In females, splenic fibrosis incidences were 6%, 29%, and 40% for control, 37.5 mg/kg rats and 75 mg/kg rats, respectively (National Toxicology Program 1990).
In female B6C3F1 mice administered glycidol via gastric intubation (25, 125, and 250 mg/kg) for 14 days, no effect on spleen or thymus weights, or leukocyte or lymphocytes counts were reported. To assess AFC response, treated mice were intravenously exposed to sheep erythrocytes on day 11 and spleen IgM AFC response was measured 4 days later. At the highest treatment dose, there was a 31% reduction in specific activity. When expressed as total spleen activity, significant decreases were noted at 125 and 250 mg/kg (29% and 41%, respectively). Splenic T-cell proliferation, in response to 10 µg/mL ConA was significantly decreased (16% and 26%, respectively) in splenocytes obtained from mice treated with 125 and 250 mg/kg glycidol. B-cell proliferation, in response to Il-4 or Il-4 and goat anti-mouse IgM F(ab')2, was only decreased in splenocytes obtained from mice treated with 125 mg/kg glycidol (13% and 16%, respectively). Comparatively, proliferation in response to goat anti-mouse IgM F(ab')2 was decreased in splenocytes from mice treated with 125 and 250 mg/kg glycidol (30-32%). While glycidol had no effect on lymphocyte blastogenesis (as assessed by splenocyte proliferative response) alone, in the presence of allogenic DBA/2 spleen cells a 25% decrease in response was noted at the middle dose only. NK cell activity in spleens was decreased at two ratios of effector:target ratios (100:1 and 50:1); the LOAELs at both ratios were 125 and 250 mg/kg, respectively. Using flow cytometry, the number and percent of B lymphocytes, T-lymphocytes, CD4 + CD8 -, CD4 -CD8 + , and CD4 -CD8cells from spleens isolated from treated mice were quantified. The total number of spleen cells, B lymphocytes, and CD4 + CD8were significantly decreased at 250 mg/kg. The LOAEL also was 250 mg/kg when the percent values of B and T lymphocytes were assessed (Guo et al. 2000).

In vitro data with cells or cell lines
To further assess the effect of glycidol on the immune function, Guo and colleagues (2000) conducted a set of ex vivo assays. Glycidol inhibited cytotoxic T cell activity in spleens obtained from mice administered glycidol via gastric intubation (25, 125, and 250 mg/kg) for 14 days. Splenocytes were sensitized with mitomycin C-exposed P815 mastocytoma cells, and co-cultured with labeled P815 cells at a variety of effector:target ratios. At an effector:target ratio of 25:1 and 0.75:1, glycidol inhibited CTL activity at a 25 mg/kg when compared to vehicle (53.8 vs. 31.5, and 8.8 vs. 2.1, respectively). At a ratio of 12.5:1, CTL activity was decreased significantly (39%) in spleens from mice treated with 125 mg/kg glycidol (Guo et al. 2000).
Resident macrophage activity (in the presence of macrophage stimulators) was assessed in mice treated with glycidol via gastric intubation (25, 125, and 250 mg/kg) for 14 days. Increased cytotoxicity was only observed after treatment with 25 mg/kg glycidol in the presence or absence of macrophage stimulators (1.7-to 2.5-fold increase) (Guo et al. 2000).
Host resistance to B16F10 melanoma cells, Listeria monocytogenes and Streptococcus pneumoniae were assessed in mice treated with glycidol via gastric intubation (25, 125, and 250 mg/kg) for 14 days. Glycidol increased pulmonary tumor formation in mice treated with B16F10 melanoma cells (LOAEL = 125 mg/kg). No effect on host resistance was noted at the three challenge levels of Listera monocytogenes (1, 2, or 4 × 10 4 CFU/mouse). At the challenge level 5.52 × 10 7 CFU Streptococcus phenumoniae/mouse, increased host resistance was observed in the 250-mg/kg glycidol treated mice (Guo et al. 2000).

Human Data
Data from epidemiology studies No data were located.

In vitro data with cells or cell lines
The effect of HC on IL-4-induced IgE production was measured in PBMCs isolated from healthy volunteers. HC induced an ~20 fold increase in IgE production at a LOEL of 1×10 -7 M. HC did not have any effect on IgE production in the absence of IL-4 (data not shown) (Nüsslein et al. 1994).
Blood samples from healthy adults were pre-treated with 30 µg/dL HC (identified as cortisol); INF production was then stimulated with Newcastle disease virus. HC decreased IFN-α response by 50-60% (data in graph) (Reissland and Wandinger 1999). Keh and colleagues (2003) reported that in septic shock patients, HC attenuated inflammatory and anti-inflammatory responses without inducing immunosuppression.

Rodent Data
Data from in vivo immunotoxicology or toxicology studies Swiss inbred mice were intraperitoneally injected with 0, 1.5, 5 or 15 mg/kg of HC. Forty-eight hours later, there was a significant decrease in thymus weight at 15 mg/kg (data in graph). To test the effect of HC on delayed hypersensitivity, mice were immunized with sheep erythrocytes in FCA, challenged in the footpad on day 5, and treated simultaneously with increasing amounts of HC. The mice received another injection of HC two hours before measuring 24-hour footpad swelling. 5 and 15 mg/kg HC suppressed footpad swelling (data in graph). Glucocorticoidinduced leukopenia and monocytopenia was evaluated in mice 2.5 hours after intravenous injection with HC. The numbers of circulating nucleated and monocytic cells was maximally decreased at the lowest dose tested (1.5 mg/kg), with the number of both cell types increasing with increasing dose (data in graph). A plasma transfer study found that 2.5 hours after transfer, the plasma of HC treated mice raised the number of nucleated cells in saline treated acceptor mice by 46%. To evaluate feedback-inhibition, mice were injected (route not specified) with 5 mg/kg HC for four days and examined 7 or 11 days (data not shown) after the last injection. At day 7, HC had no effect on delayed hypersensitivity, serum corticosterone, or numbers of circulating nucleated and monocytic cells (data in graphs) (Van Dijk et al. 1979).
In a trio of studies by El Fouhil and colleagues (El Fouhil et al. 1993a, 1993bEl Fouhil and Turkall 1993), immunologically immature rats were treated subcutaneously with 400 mg/M 2 /day HC, administered on alternate days from PND 7 to PND 19. At two days after the last treatment (PND 21), thymus and spleen weights were decreased (71 and 28%) compared to vehicle control, but at PND 42 organ weights were increased (18 and 7%). Leucocytosis was increased in PND 21 and 42 rats (12 and 24%), with a decrease in IgM concentration in serum (45 and 15%). At PND 21 there was a 46% decrease in the percentage of lymphocytes, which resolved by PND 42 (El Fouhil and Turkall 1993). On PND 21, splenic white pulp was largely depleted of small lymphocytes. There were no distinct periartiolar lymphoid sheaths and no primary follicles. The number of T cells surrounding the central arteriole was decreased (data not shown). By PND 42, the pulp appeared normal (El Fouhil et al. 1993a). On PND 21, the outer cortex of mesenteric lymph nodes was found to be depleted of small lymphocytes and primary follicles, and neither cortical expansion nor capsular indentations were detected. There was a marked depletion of B lymphocytes, which were more or less discrete and did not aggregate to form follicles. There was no apparent change in T lymphocytes. On PND 42, the lymph nodes were comparable between HC treated and control rats (El Fouhil et al. 1993b).

In vitro data with cells or cell lines
No data were located.

Mode of action information
No data were located.

Human Data
Data from epidemiology studies No data were located.

In vitro data with cells or cell lines
Heparinized whole blood, from healthy adult volunteers, was incubated with 1 to 50 µM indomethacin for 1 hour prior to stimulation with LPS. A significant increase in IL-6 expression was only noted at 50 µM indomethacin (129.7%). Comparatively, a dose-dependent increase in TNF-α was observed, and at 50 µM the number of TNF-α positive cells had doubled (204.7%) (Hartel et al. 2004).
Human PBMC were treated with 1, 10 or 100 µM indomethacin. At all tested doses, indomethacin decreased LPS-induced PGE2 synthesis to near 0%; the calculated IC50 was 0.039 µM (data in graph). Indomethacin also decreased IgG and IgM production (data in graph) at all doses tested. Indomethacin up-regulated IL-2 production and down-regulated IL-6 production in treated PBMC (data not shown). Increased PHA-, anti-CD3, and IL-2-induced lymphocyte proliferation was reported after indomethacin exposure. NK activity (against K562 target cells) was increased at 1 (1.5-fold) and 10 (1.5-fold) µM. A significant effect on LAK cell activity was not observed at 50 µM. Co-incubation of PBMCs with IL-2 and indomethacin caused an increase in IFN-γ production by LAK cells at 1, 10 or 100 µM (data not shown) (Tanaka et al. 1998).
Indomethacin (5.6 µM) increased proliferation of PHA-and ConA-stimulated lymphocytes (in mononuclear cell cultures) (data in graph). The effect was only observed at suboptimal concentrations of PHA and ConA. The observed increased proliferation was lost at optimal and supraoptimal concentrations. Additional testing showed indomethacin increased PHA-stimulated lymphocyte proliferation in a dose-dependent manner (LOEL = 0.04 µM). Removal of adherent cells from the culture negated the stimulatory effect produced by indomethacin. Indomethacin did not affect cell viability, but increased incorporation of tritiated thymidine in a dose-dependent manner (Jawad and Rogers 1984).

Mode of action information
No data were located.

Data from in vivo immunotoxicology or toxicology studies
Female C57BL/6 mice were orally administered 5 mg/kg indomethacin for 4 days. Animals were then immunized with sheep erythrocytes and then serum hemagglutination and AFC titers were assessed 4 and 8 days, or 6 days later, respectively. Indomethacin decreased both titers by approximately 40% (data in graphs). Indomethacin also decreased ConA-and LPS-induced August 8, 2018 42 stimulation of lymphocyte proliferation (data in graphs). Incubation of indomethacin (3 µM) with LPS-stimulated lymphocytes isolated from indomethacin-treated mice decreased proliferation (Barasoain et al. 1980). Female B6C3F1 mice were subcutaneously injected with 1, 2, or 4 mg/kg indomethacin for 6 days. Studies were conducted on mice 3 days after final treatment. No effect on thymus weight was reported, while 4 mg/kg indomethacin caused a 38% increase in spleen weight. A dose-dependent increase in splenic lymphocyte proliferation (10%-80%) was observed in nonstimulated cultures. Increased proliferation was observed in LPS-stimulated cultures from mice treated with 1 or 2 mg/kg indomethacin (13% and 22%, respectively), while a decrease was observed at the highest indomethacin dose. Comparatively, decreased proliferation was observed in PHA-, ConA-, or MLC-treated splenic cultures from indomethacin treated mice. Increased formation of PFC/10 6 splenocytes also were observed in treated mice (149% increase at 4 mg/kg). Indomethacin did not affect macrophage-induced inhibition of tumor cell growth (MBL-2), but did increase phagocytosis of sheep erythrocytes. Host resistance to Listeria was increased in treated mice. No effect on delayed hypersensitivity was noted .
Oral exposure to indomethacin (2.5, 5, or 10 mg/kg/day for 3 days) decreased formation of PFC in C57BL/6 mice after immunization to sheep erythrocytes. Studies showed a dose-dependent decrease in the number of PFC/10 6 spleen cells; decrease ranged from 43% to 97%. Similar inhibition was observed at 5 mg/kg/day indomethacin and various concentrations of sheep erythrocytes (2.5 × 10 8 and 5 × 10 8 ); decreases ranged from 47% to 68%. Indomethacin also inhibited antibody response to P. aeruginosa LPS; total response was decreased by 44% (Rojo et al. 1981).
Oral administration of 6 mg/kg/day indomethacin for 4 days produced a 32% decrease in total number of lymphocytes in Swiss male mice. No effect was noted at earlier time points (i.e., 2 or 3 days). An increase in the number of colonies/10 5 bone marrow cells (2.7-to 3.9-fold) also was noted in mice that were administered indomethacin for 4 days. Indomethacin also decreased PGE2 (25-43%) and PGF2α (41-56%) levels in bone marrow cells after 4 days of administration (Fontagne et al. 1980). Male CBA mice were intraperitoneally injected with 0.7, 4, or 8 mg/kg indomethacin. Two to 24 hours after exposure, mice were euthanized and spleens removed. A dose-dependent increase in splenocyte proliferation was noted after 2 hours, with a 14.3-fold increase in proliferation at the highest dose tested. A time-dependent increase in proliferation was also noted when mice were treated with 4 mg/kg indomethacin, with a maximal fold change of 31.4-fold at 24 hours. Distribution of T-cell phenotypes was not affected by indomethacin administration (Gonzalez-Cabello et al. 1987). colleagues (2007, 2009) evaluated effects of indomethacin in young Sprague-Dawley rats after in utero exposure. In 3-week old pups from dams treated with 0.25, 0.5, or 1.0 mg/kg indomethacin on GD 18-21, a significant increase (31%) in the number of spleen cells was observed in males from the highest dose group. Immunophenotyping of splenocytes showed a dose-dependent increase in the proportion of CD45RA+ cells in male pups. However, a similar increase in peripheral blood lymphocytes was noted. No effect on serum IgM or IgG levels was reported in males or females. A significant decrease in anti-KLH IgG titers, but not IgM titers was reported in males from the highest dose group tested (Kushima et al. 2007). When doses of 0.5, 1.0, or 2.0 mg/kg indomethacin were used, a significant decrease in splenocyte IL-10 levels were reported in males; no effects on IL-6, IL-2, IL-4, TNF, or IFN-γ levels were noted in either sex (Kushima et al. 2009).
Indomethacin (1 or 2 mg/kg administered twice daily for 3 days to adjuvant induced arthritic Sprague-Dawley rats) reduced PHA-induced lymphocyte proliferation in a dose-dependent manner (data in graph). LPS-stimulated proliferation was also inhibited at both doses, however the response was partially recovered at the higher tested indomethacin dose (data in graph) (Seng et al. 1990).
Indomethacin increased the total number of cells, and number of T-and B-cells up to 14 days after birth, in newborn ddy mice intraperitoneally injected with 5 µg/g every 2 days from birth (data in graphs) (Shibuya et al. 1986).

In vitro data with cells or cell lines
Indomethacin (3 µM) inhibited proliferation of lymphocytes isolated from C57BL/6 mice (data in graph). Additionally, dose-dependent inhibition LPS-induced proliferation of isolated lymphocytes was noted (Barasoain et al. 1980). Indomethacin dose-dependently increased male rat (strain not provided) ConA-induced lymphocyte proliferation after an 18-hour incubation (LOAEC = 1 µM). A time-course evaluation with 1 µM indomethacin showed that ConA-inducted lymphocyte proliferation was enhanced at incubation times up to 30 hours. Proliferation at exposure times ranging from 36 to 66 hours were not different from controls (Calder et al. 1991).
Indomethacin (50 nM to 50 µM) dose dependently increased LAK activity in BALB/c mouse splenocytes that were cocultured with recombinant IL-2. Increased lysis of JC tumor cells was observed, reaching a maximum response of 123.6 lytic units at 50 µM compared to 43.6 lytic units for IL-2 alone. Studies also showed that the increased response, compared to addition of IL-2 alone, was observed when culture conditions were maintained for up to 4 days. Addition of nylon wool to the culture, abrogated the induction of LAK response observed in the presence of indomethacin (Chao et al. 1989).
Indomethacin decreased IL-4 levels in ConA-stimulated splenocytes isolated from 3-week old male rats (LOAEL = 50 µM). No effect was noted in splenocytes from females. Decreased IL-6 splenocyte levels was observed in cells obtained from females and treated with 2.0 µM indomethacin. No effect on IL-2, IL-10, IFN-γ, and TNF-α were noted (data not shown or in graph) (Kushima et al. 2009).

Mode of action information
Indomethacin induced effects on prostaglandin synthesis was associated with several immune effects. Lala and Parhar (1988) suggested that indomethacin effects are associated with suppression of prostaglandin synthesis. Rojo and colleagues (1981) and Franceschi and colleagues (1988) proposed that indomethacin inhibition of prostaglandin synthesis leads to altered T-cell function, and NK-and antibody-dependent cytotoxicity.
Differential effects on T-cell and B-cell-induced lymphocyte proliferation were reported. A dosedependent effect on T-cell function was reported, while an inverse effect on B-cell function was noted (Seng et al. 1990).
Indomethacin has been postulated to produce immune effects through inhibition of Th1, and to a lesser extent Th2, responses (Yamaki et al. 2003). Studies conducted by Jaramillo and colleagues (1992) supported this proposed mode of action.

Data from epidemiology studies
In 19 cases of INH-induced liver failure, antibodies were present in sera of 15 patients. Anti-INH antibodies were present in 8 patients. Additionally, anti-cytochrome P450 antibodies were identified in up to 14 patients. Antibodies were not detected in patients that were treated with INH but did not have significant liver injury (Metushi et al. 2014c). In eight INH-induced liver failure patients, the dominant serum immunoglobulin isotype of anti-INH antibodies was IgG. A low titer of IgM was observed in two patients, while IgA and IgE antibodies were not detected. Phenotyping the IgG antibody indicated that the isotype was IgG3 (Metushi et al. 2014b).

In vitro data with cells or cell lines
In a series of studies, Kucharz and colleagues studies the immunomodulatory effects of IAH. In 5 µg/mL PHA-stimulated T-cells, IAH increased cellular proliferation (16% to 27%) at concentrations ranging from 0.01 to 0.0001 mM. (Kucharz and Sierakowski 1990a). In PBMC stimulated with 5 ng/mL anti-CD3 antibody, IAH produced a biphasic response. At 1 and 10 mM IAH decreased (53.6% and 24.4%, respectively) cell proliferation. Increased cellular proliferation (18-47%) was observed at concentrations ranging from 0.0001 to 0.1 mM. A similar biphasic pattern was observed when 10 ng/mL anti-CD3 antibody was used. In T-cells stimulated with anti-CD3 antibody, PHA, or PHA with PMA, IAH also modulated proliferation in a biphasic manner (Kucharz and Sierakowski 1990a). In cells stimulated with 5 µg/mL PHA and 20 ng/mL PMA, 0.1 to 10 mM IAH decreased T-cell proliferation 17% to 46%. At 0.001 mM IAH, at significant increase (21%) in T-cell proliferation was observed (Kucharz and Sierakowski 1990d). In cells stimulated with IL-2, IAH decreased cell proliferation 0.1 and 1 mM (71% and 47%, respectively) and increased proliferation at 0.01 to 0.001 mM (8% to 12%, respectively (Kucharz 1995).
IAH decreased IL-1 production from human monocytes in a dose-dependent manner in the absence or presence of lipopolysaccharide (LOAEL = 0.001 mM) (Kucharz and Sierakowski 1992).
In the absence of PHA, IAH stimulated proliferation of Jurkat cells (LOAEL = 0.01 mM). In the presence of PHA (2 or 5 µg/mL), IAH stimulation was observed at higher concentrations (1 and 10 mM) while at lower concentrations no effect was observed (Kucharz and Sierakowski 1990c).
INH (5 µg/mL) did not have any effect on the phagocytic activity or intracellular killing activity on polymorphonuclear leukocytes obtained from healthy volunteers (Okuyan et al. 2005).

Mode of action information
Metushi and colleagues proposed that INH produced an immune response that leads to liver injury (Metushi et al. 2014c(Metushi et al. , 2014b.

Data from in vivo immunotoxicology or toxicology studies
Female Nat1/2(-/-) mice were treated with INH either by oral gavage (100 mg/kg/day) for up to 7 days or by feed (0.2%) for 35 days. In mice treated by gavage, significant decrease in M1 macrophages and increase in M2a and M2b macrophages in cervical lymph nodes was noted. No effect on the M2c macrophages was observed. Comparatively, no effect was noted in the macrophage phenotypes obtained from mice that were exposed by feed (Metushi et al. 2014a).
INH (0.1 to 1.0 mg/10 µL) did not alter the weight of popliteal lymph nodes from C57BL/10 mice 7 days after subcutaneous injection (Kammuller et al. 1989). A lack of effect on popliteal lymph nodes from Brown Norway rats also was observed when exposed to 5 mg/50 µL INH (Verdier et al. 1990).

In vitro data with cells or cell lines
In HT-2 cells, stimulated with IL-2 (3 or 30 U/mL), increased proliferation was observed at 1 and 10 mM IAH at 30 U/mL and only at 1 mM at 3 U/mL. No effect on proliferation was observed in cells stimulated with 60 U/mL IL-2 (Kucharz and Sierakowski 1990c). Additionally, no effect on proliferation by IAH was observed in HT-2 cells stimulated with PMA (data not provided) (Kucharz and Sierakowski 1990d).

Mode of action information
No data were located.

Human Data
Data from epidemiology studies No data were located.

In vitro data with cells or cell lines
Lead (lead acetate 5.0 mg -1.5 ng/mL, or lead chloride 0.5 mg -0.15 ng/mL for 24 hours) significantly reduced cell vitality and/or proliferation and affected secretion of proinflammatory, TH1 and TH2 cytokines in human peripheral mononuclear blood cells that were stimulated with either heat-killed Salmonella enteritidis (hk-SE) or monoclonal antibodies. At lower lead levels, expression of IFN-γ, IL-1β and TNF-α were reduced. Monoclonal antibody induced IL-4, IL-6 and IL-10 and hk-SE induced IL-10 and IL-6 levels were increased in the presence of lower lead levels. The authors suggest that lower dose lead suppresses the TH1 cytokine and the proinflammatory cytokines while the increased IL-4 and/or IL-10 production can induce and maintain a TH2 immune response (Hemdan et al. 2005).
Thirty male lead-exposed (battery recycling industry) workers with a blood lead level > 10 µg/dL and 27 unexposed healthy volunteers without any history of occupational exposure to lead were selected for this study. The serum level of IgA was found to be significantly increased in the lead-exposed group as compared to controls. No differences were observed in serum IgG and IgM levels. Both the level of nitric oxide production after stimulation with zymosan-A and the neutrophil respiratory burst as measured by nitroblue tetrazolium reduction were comparable in neutrophils from lead-exposed and unexposed volunteers (Mishra et al. 2006).

Mode of action information
Lead acetate (1 µM) induced activation of NF-κB in primary human CD4+ T lymphocytes. This lead induced activation was blocked by antibodies for p65 and p50 subunits (indicating that the p65:p50 heterodimer (NF-κB) is involved), but not by cRel. Lead acetate (100 pM -100 µM) did not activate NF-κB in 4 different T cell lines, suggesting that these cell lines may not be a reliable system for studying transcriptional activation in human T cells (Pyatt et al. 1996).

Data from in vivo immunotoxicology or toxicology studies
Lead acetate suppressed macrophage-dependent immune responses in cells taken from female BDF1 mice exposed to lead in drinking water at concentrations from 0 to 1000 ppm for 3 weeks.
The T-cell dependent sheep erythrocyte primary immune response was suppressed by approximately 40-50% in all lead-exposed groups. In contrast, the E. coli lipopolysaccharide (Tcell and macrophage-independent) induced response, was not suppressed (Blakley and Archer 1981). Lead did not alter the ability of T-cell mitogens to induce interferon (Blakley et al. 1982).
Lead acetate effects were modulated by maternal protein intake. Fischer 344 rats were exposed to lead acetate (250 ppm) in the drinking water during breeding and pregnancy until parturition and were fed isocaloric diets (either 20% casein or 10% casein). Offspring exposed to lead and high maternal dietary protein had significantly elevated levels of both IL-4 and TNF-α (values not provided). Offspring exposed to lead and low maternal dietary protein had significantly reduced IL-4 levels compared to the lead control group (values not provided). No other changes were observed, and immune parameters measured in the dams were not affected by treatment (Chen et al. 2004).
In a study comparing immunotoxic effects of various lead salts, Balb/c mice were treated for five consecutive days between immunization and elicitation with intraperitoneal injections of 0.5 or 6 mg/kg of a lead salt. A statistically significant increase in delayed hypersensitivity (as measured by footpad swelling) was observed following administration of lead acetate (55.0% increase in footpad thickness as compared to controls; LOAEL = 6 mg/kg) (Descotes et al. 1984).
Exposure to lead acetate resulted in a decreased ability of mice to survive a sublethal dose of a virulent strain of S. typhimurium. C3H/HeN mice were exposed to lead acetate (5 or 10 mM) in the drinking water for up to 18 weeks. At week 16, mice were infected with S. typhimurium. 40% of the mice exposed to 5 mM lead acetate survived the infection with a median survival of 26 days. None of the mice treated with 10 mM lead acetate survived, with death occurring within three weeks of becoming infected. In contrast, 80% of control mice survived with a median survival of 60 days. The ability of splenocytes, cultured from the lead-treated and control mice showed a marked reduction in the production of IFN-ℽ (27% and 35% in mice treated with 5 and 10 mM lead acetate, respectively) and IL-12p40 (42-45% in mice treated with 5 and 10 mM lead acetate, respectively, as compared to induced control). Secretion of IL-4 by splenocytes from leadtreated mice was 3 to 3.6-fold higher than in control mice (Fernandez-Cabezudo et al. 2007).
Adult Sprague-Dawley females were treated with 500 ppm lead acetate via drinking water either early in gestation (days 3-9) or late in gestation (days 15-21). Offsping were assessed as adults. Significantly depressed DTH responses as well as increased IL-10 production, relative monocyte numbers and relative thymic weights were reported in female offspring exposed to lead during late gestation. Male offspring exposed during late gestation had significantly increased IL-12 production and decreased IL-10 production while the DTH response, relative monocyte numbers and thymic weights were unchanged compared to controls. The authors found that adherent splenocytes (likely macrophages) and T lymphocytes are the primary immune cells affected during fetal lead exposure and that gender may influence immunotoxicity due to lead exposure (Bunn et al. 2001).
Lead acetate increased IL-4 production in mice at 40 and 400 mg/L and decreased IFN-γ levels in mice at 400 mg/L. Adult Swiss mice were administered lead acetate in drinking water for 14 days. The authors concluded that low level lead exposure enhances a Th2 response while high lead levels can either stimulate Th2 immune activity or reduce Th1 activity, thus resulting in an imbalance between Th1 and Th2 activation (Iavicoli et al. 2004).
Lead acetate (100 or 1000 ppm in drinking water) did not alter the ability of splenocytes isolated from exposed male Alderly Park rats to mediate native and interferon activated natural cytotoxicity at 2,4,6 and 8 weeks following commencement of exposure. Splenic T-cell function of treated rats as determined by phytohaemagglutinin induced proliferation was comparable to control values (Kimber et al. 1986).
Lead acetate (200 ppm either in the drinking water or given intraperitoneally for 4 weeks) decreased the number of lymphocyte cells and cellularity (i.e., number of cells per mg tissue) in the thymus, but no significant changes in either parameter were reported for the submaxillary lymph nodes. Proliferation of T cells stimulated by ConA and proliferation of B cells stimulated by LPS was increased by lead in the thymus by both routes of exposure. In the submaxillary lymph nodes, there was a decrease in the proliferation of T cells following treatment by either route (Teijon et al. 2010).

In vitro data with cells or cell lines
RAW 264.7 cells were treated with 100 ppm lead acetate for 24 hours in the presence or absence of LPS. Lead produced a statistically significant inhibition of the level of LPS-induced nitric oxide (data not provided). No effect on cytotoxicity was observed (Mishra et al. 2006).

Mode of action information
C3H/HeN mice were exposed to lead acetate (0, 5 or 10 mM in drinking water for periods of up to 18 weeks) and inoculated with a virulent strain of S. typhimurium. Sera were collected on days 15 and 38 post infection. The authors report that the IgG2a antibodies were elevated in control mice by day 38 post infection (0.09 ± 0.05 on day 15 vs. 0.30 ± 0.03 on day 38; an increase of 300% from day 15), but were only slightly increased in lead-exposed mice (0.11 ± 0.01 on day 15 vs. 0.16 ± 0.02 on day 38). IgG1 isotype antibodies (an isotype induced by IL-4) were significantly elevated in lead exposed mice on day 38, as compared to control mice. The authors conclude that lead acetate induces a subtle but substantial shift toward a Th2-type immune response to infection with Salmonella organism (Fernandez-Cabezudo et al. 2007).
A single intraperitoneal exposure to lead acetate (12 mg/kg) in B6C3F1 mice produced changes in cell surface markers on discrete subpopulations of lymphoid cells from the spleen and bone marrow. The authors concluded that while the changes may not correlate with functional activity of the cells, they seemed to predict a shift to immature cell types, which correlated with the increase in progenitor cells observed (Burchiel et al. 1987).

Human Data
Data from epidemiology studies No data were located.

In vitro data with cells or cell lines
Increased urinary excretion of 9α,11β-prostaglandin F2 and leukotriene-4 were reported in association with mannitol-induced bronchoconstriction in 14 asthmatic patients. Urinary excretion of 9α,11β-prostaglandin F2 and leukotriene-4 increased from 61 to 92 and 19 to 31 ng × mmol/creatinine, respectively (Brannan et al. 2006). A separate study reported that repeated challenge with mannitol induced refractoriness in asthma patients. The mannitol refractoriness was associated with maintained release of 9α,11β-prostaglandin F2 and leukotriene-4 (Larsson et al. 2011).
Increased proportion of submucosal MCTC was observed in asthmatic individuals with airway hyperresponsiveness to mannitol compared to asthmatic individuals without responses to mannitol. The percentage MCTC increased from 18.7% to 40.3%, but the increase in the numbers of MCTC between the two groups was not significantly increased. Increased gene expression of thymic stromal lymphopoietin and carboxypeptidase AM also were reported (Sverrild et al. 2016).
Mannitol did not induce DNA damage in human leukocytes at concentrations from 1.25 to 10 mM (Frenzilli et al. 2000).
Mannitol (22 mmol/L) did not increase IL-6 or TNF-α secretion from monocytes treated with glucose (11 mmol/glucose) for 24 hours. A similar lack of effect was observed when cells were incubated for 48 hours (Morohoshi et al. 1996).
Mannitol did not inhibit growth of human granulocyte precursor cells at a concentration up to 5 mM (Holdener et al. 1983).

Mode of action information
Mannitol is shown to narrow the airway in asthmatic, but not healthy, test subjects (Brannan et al. 2001(Brannan et al. , 2003(Brannan et al. , 2000. Mannitol is proposed to increase osmolarity of airway surface liquid, leading to an increase in mediator release (e.g., histamine, prostaglandins, and leukotrienes) from inflammatory cells which induces bronchoconstriction (Brannan et al. 2006;Sverrild et al. 2016). One mediator that is proposed to be released is prostaglandin D2 from mast cells (Brannan et al. 2003(Brannan et al. , 2006.

Data from in vivo immunotoxicology or toxicology studies
No data were located.

In vitro data with cells or cell lines
No data were located.

Mode of action information
No data were located.

Human Data
Data from epidemiology studies No data were located.

In vitro data with cells or cell lines
In PBMC from five nickel allergic individuals 0.1 mM NiSO4 increased IL-4 and IFN-γ production. The peak effect was lower than when PBMC were incubated with PHA (data shown in graph) (Thomas et al. 2003).

Mode of action information
NiSO4 was shown to alter dendritic cell phenotypes by activation of MAPKs and NF-B. Additionally, NiSO4 induced IL-8, IL-6, and IL-12 p40 production (Ade et al. 2007;Antonios et al. 2009). Activation of the MAPK pathway may lead to upregulation of the Cys-Cys chemokine receptor, CCR7, which allows dendritic cells to migrate to the draining lymph nodes (Boisleve et al. 2004).
NiSO4 has a similar capacity to stimulate polyclonal CD+4 in Ni-allergic and -nonallergic individuals. Differences in clonal expansion or presence of Ni-binding motifs in MHC class II complexes could be involved in the development of allergic contact dermatitis (Lisby et al. 1999).

Data from in vivo immunotoxicology or toxicology studies
Male Wistar rats were intratracheally instilled with 1, 2, 4, or 8 µmole NiSO4 per rat. The rats were euthanized up to 7 days after treatment. Treatment decreased the percentage of lymphocytes in pulmonary lymphoid cells (~55% to ~40%). NK activity in lymphoid lung cells was dependent on concentration and effector:target cell ratio. NK activity was decreased 1 day after treatment of 4 and 8 µmole NiSO4 at the effector to target cell ratio of 6:1. Two days after treatment suppression of NK activity was significant at doses ≥2 µmole NiSO4 and at the effector to target cell ratio of 6:1. After 7 days, a significant decrease was only observed at 8 µmole. NiSO4 did not significantly modulate alveolar macrophage cytotoxic activity towards 3T12 target cells. Decreased levels of TNF-α was reported at all time points, while increased IFN-γ level was only noted after exposure to 8 µmole/rat on day 2 (data in graph) (Goutet et al. 2000).
Female B6C3F1 mice were exposed to NiSO4 aerosol for 6 hours per day, 5 days per week for 65 days. The actual exposure concentrations tested were 0.027, 0.11, or 0.45 mg Ni/m3. No change in thymic weight was reported. A significant increase in the number of nucleated cell numbers from lung-associated lymph nodes (LALN) and lavage fluid, after mice were immunized with sheep red blood cells, was noted at the highest dose tested (1.72-and 3.86-fold, respectively). Nonsignificant increase in the total antibody-forming cells (AFC)/(LALN) and nonsignificant decrease in AFC/spleen, after immunization with sheep red blood cells, were also noted after NiSO4 exposure. NiSO4 had no effect on mixed lymphocyte response of spleen cells after exposure to mitomycin C-treated spleen cells from DBA/2 mice. No effect in mitogenstimulation assays also were noted by NiSO4 exposure. NiSO4 modulated pulmonary alveolar macrophage function, as measured by phagocytosis of opsonized erythrocytes; activity was significantly increased at 0.11 mg Ni/m3 (data not provided). Comparatively, NiSO4 had no effect on peritoneal macrophage phagocytosis activity at any tested dose. The highest dose of NiSO4 was associated with a significant two-fold increase in the number of B16F10 tumor nodules in the lungs of treated animals. However, incorporation of radiolabeled uridine was not considered biologically significant. NiSO4 did not affect splenic NK cell cytolytic activity (Haley et al. 1990).
Histopathological lesions in lungs, liver, thymus, kidneys, spleen, and lymph nodes were noted in male F344 rats intramuscularly injected with 125 µmole/kg NiSO4 over 26 days. Thymus glands from rats treated with the highest dose were much smaller than controls. Corticomedullary junction was not distinct and extensive degeneration and depletion of lymphocytes in the thymic cortex were noted. Additional tissues from these rats were evaluated further. In the lungs, large alveolar macrophages and polymorphonuclear leukocytes were noted in alveolar spaces and exudate. In the spleen and lymph nodes, lymphocytes were focally depleted in the white and red pulp (Knight et al. 1991).
Male Sprague-Dawley rats were exposed to 0.02, 0.05, and 0.1% NiSO4 in drinking water for 13 weeks. Effects on splenic lymphocyte and thymocyte subpopulations were evaluated. In splenic lymphocytes, increases in the total number of T-cells (LOAEL = 0.05%) and CD8+ T-cells (LOAEL = 0.02%) were reported. For CD4+ T-cells, the number of cells increased at 0.05% NiSO4 and then decreased at 0.1% dose. An increase in the total number of B cells was noted at 0.05% NiSO4. Subchronic exposure to 0.02% NiSO4 also increased the percentage and absolute number of thymocyte CD8+ cells. Exposure to 0.05% NiSO4 increased the total number of thymocyte cells, the percentage and absolute number of CD8+ cells, and absolute numbers of both CD4+ and B-cell populations. Exposure to 0.1% NiSO4 decreased the total number of thymocytes, the percentage and absolute number of CD4+ T cells, and absolute numbers of CD8+ T cells and of B cells (Obone et al. 1999).
Male C3H/He mice were provided 0.01, 0.05, 0.1, 0.25, 0.5, or 1% NiSO4 for 7 or 10 weeks. Mice were then sensitized with NiSO4 for 7 days and the footpad thickness was measured. The mice were then challenged with 0.4% NiSO4 and footpad swelling was measured 24 hours later. After 7 weeks of oral exposure, footpad swelling was not reduced at any of the tested doses.
Lymph nodes from C3H/He mice sensitized to NiSO4 were incubated with various monoclonal antibodies and then injected into naïve mice. After challenging with NiSO4, footpad swelling was measured. Cells treated with CD4-, Thy1.2-, or Ig-specific antibodies showed reduced swelling while cells treated with CD8 antibodies induced footpad swelling (Ishii et al. 1993).
Macrophage and PMN chemotactic activities in bronchoalveolar fluid were increased at 2 days after intratracheal instillation of 50 µg Ni per male Wistar rat. Activity then decreased until end of the experiment (14 days). Comparatively, LTB4 were maximally decreased at day 1 and then increased to control levels by day 14 (Hirano et al. 1994).

In vitro data with cells or cell lines
Spleen cells from C57BL/6 and Rag-1 deficient mice were stimulated with varying concentrations of NiSO4 (concentrations not provided). Using the ELISPOT assay, IL-2, IL-4 and IFN-γ secreting cells were identified in splenic cells from C57BL/6 mice. The number of IFN-γ cells were greater than the IL-2 and IL-4 cells. At higher concentrations (≥400 µM), the numbers of IL-2 and IL-2 secreting cells decreased while those secreting IFN-γ remained high. The number of IFN-γ cells did not increase due to previous immunization of NiSO4. In splenic cells from Rag-1 deficient mice, NiSO4 also contained IFN-γ secreting cells. However, at higher concentrations the cell levels decreased (in comparison to wild-type). Addition of NK1.1 antibodies produced a partial depletion in the cells. Further studies showed that addition of NKG2D antibodies reduced the number of IFN-γ secreting cells in wild-type and RAG-1 deficient mice (Kim et al. 2009).

Mode of action information
No data were located.

Human Data
Data from epidemiology studies No data were located.

In vitro data with cells or cell lines
No data were located.

Mode of action information
No data were located.

Data from in vivo immunotoxicology or toxicology studies
Female B6C3F1 mice were exposed to 30, 100, or 300 mg/kg nitrobenzene for 14 days via gastric intubation. Hepatomegaly and splenomegaly were observed in mice that received 100 and 300 mg/kg nitrogenzene. Mild congestion in the red pulp areas of the spleen was noted in mice that received 100 mg/kg, while the spleen was dark red in those that received 300 mg/kg. Absolute and relative spleen weight were significantly increased (LOAEL = 100 mg/kg). Comparatively, absolute and relative thymus weights were increased only at 100 mg/kg. The number of bone marrow cells increased in a dose-dependent manner (LOAEL = 30 mg/kg). At the highest dose tested the increase was 60% above controls. DNA synthesis and the number of CFU-GM per femur also were increased (LOAELs = 30 mg/kg). In response to sheep erythrocytes, a significant increase in spleen weight (62%) and spleen cell number (29%) was observed at 300 mg/kg, when animals were sensitized four days after nitrobenzene exposure. Comparatively, a decrease in IgM AFCs were decreased (LOAEL = 100 mg/kg). When responses to sheep erythrocytes were observed (sensitization occurred 5 days after nitrobenzene exposure), spleen weight and cells were increased at 100 and 300 mg/kg. However, no effects on IgG AFC were noted. When 20 days lapsed between nitrobenzene exposure and sensitization to sheep erythrocytes, no effects were reported. No effect on delayed hypersensitivity was reported at any of the tested doses. Splenic proliferation responses induced by PHA and ConA were suppressed by exposure to nitrobenzene (LOAEL = 100 mg/kg). No effect on LPS-induced proliferation were reported. Responses to DBA/2 mice spleen cells also were decreased (LOAEL = 100 mg/kg). Using radiolabeled sheep erythrocytes, the phagocytic index was shown to be increased in a dose-dependent manner. The phagocytic activity of peritoneal cells also was increased in a dose-dependent manner (LOAEL = 300 mg/kg). The ability of spleen cells to lyse radioactivity from YAC-1 target cells also was evaluated. Nitrobenzene exposure produced a decrease in lysis capacity at 100 and 300 mg/kg at effector:target ratios of 100:1 and 30:1.
Nitrobenzene did not affect host resistance to Streptococcus pneumonia, Plasmodium berghei, herpes simplex 2, or B16F10 melanoma. Comparatively, host resistance to Listeria monocytogenes was decreased. A challenge of 6 x 10 3 L. monocytogenes per

Human Data
Data from epidemiology studies No data were located.

In vitro data with cells or cell lines
No data were located.

Mode of action information
No data were located.

Rodent Data
Data from in vivo immunotoxicology or toxicology studies B6C3F1 mice were orally administered 100, 300, or 500 mg/kg BCP for 14 days. No effect on spleen or thymus weight were reported. No effect on delayed hypersensitivity response (to keyhold limpet hemocyanin), antibody response to sheep erythrocytes, serum IgM, IgA, or IgG levels, or splenic lymphocyte proliferation were noted. Absolute and relative liver weights were increased at the highest dose group. Additionally, BCP-treated mice did not develop tumors after challenge with PYB6 tumor cells (vs. controls which had a 15% tumor incidence) (Birnbaum et al. 1986).
BCP produced contact hypersensitivity in female B6C3F1 mice (Stern et al. 1991).

In vitro data with cells or cell lines
No data were located.

Mode of action information
No data were located.

Data from epidemiology studies
Several studies have suggested that prenatal PFOA exposure is linked to immunosuppressive and immunotoxicant effects observed in offspring. Granum and colleagues (2013) reported that maternal PFOA blood levels, collected at birth, were positively associated with decreased rubella antibody-levels (β = -0.40) and an increased number of common cold episodes in children from 0-3 and 2-3 years old. Cord blood IgE levels also were suppressed in female infants with high maternal PFOA levels. However, no effects on number of 18 month-old infants with allergies (e.g., food allergy, eczema) or infections (e.g., otitis media, pneumonia, skin infections, chicken pox) were noted (Okada et al. 2012). Okada and colleagues noted that while the correlation between fetal PFOA levels and the evaluated endpoints were not available, the results suggest that PFOA produced immunosuppressive effects after prenatal exposure.
A positive association between serum PFOA in adults and development of ulcerative colitis also was reported. However, a positive association with other autoimmune diseases, such as Type 1 diabetes, lupus, multiple sclerosis, Chron's disease, and rheumatoid arthritis, was not observed (Steenland et al. 2013).
Chang and colleagues (2016) conducted a systematic review to summarize and evaluate epidemiological literature on PFOA and perfluorooctanesulfonate (PFOS) with relation to evaluated immune endpoints. Endpoints evaluated included immune biomarker levels (e.g., IgE levels, white blood cell count, and C-reactive protein), immune gene expression patterns, atopic or allergic disorders (e.g., asthma, eczema, and food allergy), infectious disease (e.g., common cold), vaccine response, and autoimmune and inflammatory conditions (e.g., ulcerative colitis, rheumatoid arthritis, and osteoarthritis). The authors stated that the totality of the data limited development of a conclusion on the causal relationship between PFOA and/or PFOS exposure and evaluated endpoints due to inconsistent results and confounding factors.

Mode of action information
Direct modulation of NF-B has been implicated in modulation of cytokine production and secretion (Corsini et al. 2012). PFOA interaction with the PPARα receptor also was implicated in immunomodulatory effects in human cells. Receptor interaction was associated with reduced p65 phosphorylation and NF-B-mediated transcription (Corsini et al. 2011). The extent the role of PPARα receptor activation plays in human effects is unclear given the low level of human receptor expression (Corsini et al. 2014).

Data from in vivo immunotoxicology or toxicology studies
Animal studies suggest that PFOA exposure can affect innate and adaptive immune functions in vivo. Dietary exposure to PFOA (0.02% w/w) for 7 days significantly decreased spleen and thymus weight, and splenocyte and thymocyte levels in wild-type C57Bl/6 mice. Spleen weight and splenocyte numbers were not affected in PPARα-null mice (Yang et al. 2002). Dose response studies in C57Bl/6N mice reported that PFOA decreased absolute and relative spleen weights (LOAEL = 7.5 and 15 mg/kg/day, respectively) and absolute and relative thymus weights (LOAEL =15 mg/kg/day for both endpoints). Organ weight effects were generally reversed by 15 days after exposure was terminated (DeWitt et al. 2008). No effect on organ weights was reported in PPARα knockout mice treated with 7.5 or 30 mg/kg/day PFOA for 15 days (DeWitt et al. 2016).
PFOA exposure in drinking water was associated with reduced IgM antibody titers in C57Bl/6J and C57Bl/6N mice (DeWitt et al. 2008(DeWitt et al. , 2016. Removal of the adrenal glands in C57Bl/6N mice did not reverse reductions in IgM antibody titer levels, suggesting that the observed suppression was not in response to corticosterone production (DeWitt et al. 2009).
Modulation of the complement system was observed in C57Bl/6 mice administered PFOAtreated diets. In mice provided diets containing PFOA for 10 days, activity of the classical and alternative pathways of the complement system was decreased (N/LOAEL = 0.01%/0.02%, respectively). Serum C3 levels also was decreased by PFOA (N/LOAEL = 0.01%/0.02%, respectively). Results showed that PFOA-induced hepatotoxicity was associated with activation of the complement system (Botelho et al. 2015).

In vitro data with cells or cell lines
Reduced lymphocyte proliferation was observed in cells isolated from C57Bl/6 mice treated with diets containing 0.02% PFOA for 7 days. No effect was observed in lymphocytes isolated from PPARα-null mice also provided diets containing 0.02% PFOA (values not provided) (Yang et al. 2002). Increased ex vivo production of TNF-α in cells isolated from peritoneal cavity (2.2-fold) and bone marrow (1.7-fold), and IL-6 in cells isolated from peritoneal cavity (2.6-fold) was observed in mice treated with 0.02% dietary PFOA for 10 days. Comparatively, TNF-α production was decreased (0.8-fold) in cells isolated from spleen of treated animals (Qazi et al. 2009). IgM or IFN-γ production levels were not modulated in intrahepatic immune cells isolated from male C57Bl/6 mice provided diets with 0.002% (w/w) PFOA for 10 days (Qazi et al. 2010).
Ex vivo co-cultures of splenic CD4+CD25+ and CD4+CD25-T cells offspring gestationally and lactationally exposed to PFOA were assessed for effects on IL-10 production. Results showed IL-10 produced was significantly decreased at all doses 61%-75% in cells obtained from male offspring (LOAEL = 0.02 mg/kg). Ex vivo measurement of autoreactivity antibodies in female mice gestationally and lactationally exposed to 0.02 and 2 mg/kg PFOA showed an decrease (26%) in anti-ssDNA (Hu et al. 2012).

Mode of action information
PFOA suppresses T-cell-dependent and T-cell-independent antibody responses . The role of PPARα in PFOA-induced immunosuppression may be strain dependent (Corsini et al. 2014). PFOA-induced effects on humoral immunity may occur through effects on B-cell/plasma cell function (DeWitt et al. 2016). Direct effects on immune cells also are a proposed mode of action of PFOA (Corsini et al. 2014).
The lack of impact of removal of the adrenal gland on PFOA-induced inhibition of IgM antibody titer levels suggests that the observed effects are not dependent on elevated corticosterone levels in mice (DeWitt et al. 2009).
Effects on lymphoid organ weights and measures of immune function (i.e., thymus and spleen) indicate that they are differentially sensitive to PFOA effect. The biological basis for this difference is not known (DeWitt et al. 2016).

Data from epidemiology studies
No difference was noted in lymphocyte counts between individuals with or without toluene exposure (Akbas et al. 2004).

In vitro data with cells or cell lines
No data were located.

Mode of action information
No data were located.

Data from in vivo immunotoxicology or toxicology studies
Male C3H mice were exposed to 9 ppm toluene (nose-only inhalation exposure) for 30 minutes on study days 0, 1, 2, 7, 14, 21, and 28. Mice also were immunized with ovalbumin. Toluene exposure significantly increased total cell (3-fold) and macrophage (3.1-fold) count in BAL 24 hours after final exposure. No effect on lymphocyte count was noted. BDNF level in BAL was increased in toluene-exposed mice that were immunized with ovalbumin (data in figure). Splenic ratio of CD4 and CD8 cells in control and toluene-exposed mice were not significantly different; 3.95 and 4.14, respectively. Treatment with ani-CD4 antibody decreased the ratios to 0.65 and 0.49, respectively. Toluene exposure significantly increased plasma levels of nerve growth factor (data in figure), but did not increase plasma BDNF levels (data not provided) (Fujimaki et al. 2009).
Male C57BL/10 and B10.BR/Sg mice were inhalationally exposed to 0, 5, and 50 ppm toluene for 6 hours per day, 5 days per week for 6 weeks. Subgroups of control and treated mice were administered ovalbumin prior to exposure. Toluene exposure did not impact ConA-or LPSinduced proliferation of spleen cells from C57BL/10 mice. While no effect of ConA was noted in B10.BR/Sg (not treated with ovalbumin) mice spleen cells, 50 ppm toluene significantly increased the LPS-induced proliferation of spleen cells. Comparatively, 50 ppm toluene significantly decreased spleen cell proliferation in B10.BR/Sg mice treated with ovalbumin (data in graphs). Toluene did not alter expression of CD3, CD19, and CD11b (data not provided). Forkhead box P3 (Foxp3) transcription was significantly increased in spleen cells from B10.BR/Sg mice exposed to 5 ppm toluene and ovalbumin, when compared to controls and those not treated with ovalbumin. No effect on GATA3 or T-bet expression was noted (Fujimaki et al. 2010).
Pregnant C3H/HeN mice were exposed to 50 ppm toluene via inhalation on GD 14-18. Additionally, male offspring of unexposed dams were exposed to 50 ppm toluene on PND 2-6 or 8-12. The following table summarizes the effects observed in male offspring on PND 21. Pregnant C3H/HeN mice were exposed to 5 or 50 ppm toluene via inhalation on GD 14-18. Additionally, male offspring of unexposed dams were exposed to 5 or 50 ppm toluene on PND 2-6 or 8-12. In the hippocampus of PND 21 male offspring, TNF-α and NF-κB mRNA were significantly increased in mice exposed to 50 ppm on PND 2-6 when compared to controls (data in graphs). TNF-α, CCL3, and NF-κB were increased in mice exposed to 5 ppm on PND 8-12 (data in graphs) (Win-Shwe et al. 2012b).

In vitro data with cells or cell lines
Toluene (500 µM) exposure significantly increased ConA-(1.8-fold) and LPS-(2.1-fold) induced proliferation of spleen cells from female C57BL/6 mice. However, at the same concentration toluene did not modulate NK activity or suppress CTL formation (Grayson and Gill 1986).

Mode of action information
Low-level (5 ppm) inhalational exposure to toluene activates the STAT6, STAT5, and Foxp3 signaling pathway to enhance Th2-related and Treg-related responses in B10.BR/Sg mice treated with ovalbumin (Fujimaki et al. 2010). Toluene also enhanced NF-B, STAT5, and NF-AT in thymus cells of C3H/HeN mice inhalationally exposed to toluene (Liu et al. 2010).
Toluene modulation of IL-2 synthesis, after oral exposure, may play a role in observed immunotoxic effects (Hsieh et al. 1989). In vitro data with cells or cell lines TBTC dose-dependently decreased the percentage of colony forming unit-granulocyte macrophage (CFU-GM) colonies at concentrations ranging from 0.001 to 3.3 µM (data not provided) ).

References
Cytokine release from human lymphocytes was assessed in whole blood obtained from three donors. IFN-γ was evaluated in blood from two donors after PHA stimulation for 72 hours. Comparatively, TNF-α was evaluated in blood from three donors after LPS stimulation for 72 hours. Overall, IFN-γ and TNF-α was modulated (i.e., either increase or decrease release) in all tested samples .
Long-term cultures of human bone marrow cells were incubated with 0.001 µM TBTC in the presence or absence of a cytokine mixture for 7 or 14 days. A significant decrease in the percentage of CD19+CD22+ cells, in the absence of effects on the total lymphocyte population or percentage of T-cell subsets was reported after 7 and 14 days. Addition of cytokine mixture had no effect on TBTC effects. TBTC also induced cell death in CD19+ lymphocytes, in the absence of PPAR-γ receptor expression (Carfi et al. 2010).

Mode of action information
In vitro toxicogenomic studies in Jurkat cells (human lymphoblastic T-cell line) showed that TBTC activated cellular stress response and retinoic-acid mediated response genes (Shao et al. 2013).

Data from in vivo immunotoxicology or toxicology studies
In a 2-week study, male Wistar rats were provided diets containing 15, 50, or 150 ppm TBTC. A dose-related decrease in relative and absolute spleen and thymus weights were reported (LOAEL = 50 and 15 ppm, respectively). Concurrent to the change in thymus weight, a decrease in thymic cell counts also was observed (LOAEL = 50 ppm). However, no signs of increased lymphocyte destruction in the spleen was observed. A dose-related increase in relative liver weight was reported (LOAEL = 50 ppm). Decreased thymus weight also was observed in rats fed 100 ppm TBTC for 4-weeks (43% of control weight); no effects on spleen or liver weight were noted (Snoeij et al. 1985).
In utero and lactational exposure effects of TBTC (0.025, 0.25, or 2.5 mg/kg/day) were evaluated in Sprague-Dawley rats. Dams were orally dosed with TBTC from GD 8 until weaning. After weaning, pups were orally exposed to the same dose as the dam until sacrifice (up to PND 90). In males, a significant decrease in spleen weight was only observed in pups treated with 0.25 mg/kg/day on PND 30. A significant decrease in thymus weight also was noted on PND 30 (LOAEL = 2.5 mg/kg/day) . Serum IgM levels were increased in 30-and 60day old female offspring, while IgA, IgM, IgG, and IgG2a levels were increased in 90-day old male rats (Tables 1 and 2) ). 73. 0 ± 15. 0 †Values provided as pg Ig/mL serum × 10 4 (standard error of the mean ± standard error). 13.6 ± 1.7 20.6 ± 3.6 41.0 ± 19.4 20.9 ± 2.2 >0.05 †Values provided as pg Ig/mL serum × 10 4 (standard error of the mean ± standard error). * Significantly different from control.
The number and percentage of NK cells was increased in 30-day female and male offspring (LOAEL = 2.5 mg/kg/day). A dose-dependent increase in the number and percentage of NK cells also was noted in 90-day male rats. In 60-day female offspring an increase in the percentage of CD4+8+ T lymphocytes (LOAEL = 0.25 mg/kg/day). No anti-sheep erythrocyte IgM response or lymphoproliferative activity of splenocytes in response to mitogen stimulation was noted in 60-day old female rats or 90-day old male rats (data not provided). Delayed-type hypersensitivity to oxazolone was increased in 90-day old male rats at 0.025 and 0.25 mg/kg/day and decreased at 2.50 mg/kg/day. Mean colony forming L. monocytogenes bacteria was non-linearly increased at 48 hours post-infection and statistically significant in pairwise comparisons (0.25 mg/kg/day) in 60-day old females. In 90-day old males, a non-linear dose-response trend 3 days after infection was reported. No effects in serum levels of IL-2, TNF-α, IFN-γ, and IL-1β were reported in males or females. A non-linear dose-response increase in NK activity in 60-day females was reported .
Lactational exposure in mice to TBTC also impaired innate immunodefenses in offspring. C57BL/6 pregnant mice were given drinking water with 15 or 50 µg/mL TBTC from parturition to weaning. Clearance of Escherichia coli K-12 from the peritoneal cavity and spleen of offspring treated with 15 µg/mL TBTC was significantly decreased (Kimura et al. 2005).
ICR mice were orally dosed with 0.5, 4, or 20 mg/kg TBTC for 28 days. Relative spleen and thymus weights were significantly decreased at the highest dose tested (46% and 59% decrease, respectively). TBTC also decreased the number of plaque forming cells in response to exposure to sheep red blood cells (LOAEL = 4 mg/kg). TBTC also suppressed delayed-type hypersensitivity response to sheep red blood cells when assessed 24 and 48 hours after injection (LOAEL = 4 mg/kg). TBTC suppressed T-lymphocyte proliferation in a dose dependent manner (LOAEL = 20 mg/kg). Increased percentage of early-and late-stage thymocyte apoptosis, and expression of Fas protein expression in proteins also were noted (LOAEL = 4 mg/kg) (Chen et al. 2011).
Esophageal tubing of male C3H/Hen mice with 10 or 100 ppm TBTC for 1 week was associated with decreased NK activity. NK activities were inhibited 36% to 46% at effector:target (YAC-1 cells) ratios of 25:1 and 50:1, respectively. A significant decrease in the percentage of large granular lymphocytes (~60%) also was noted 1 week after end of treatment (Ghoneum et al. 1990).

In vitro data with cells or cell lines
Neutrophils and macrophages from mice lactationally exposed to TBTC (15 or 50 µg/mL) were isolated from peritoneal exudates. Bacterial binding to isolated neutrophils from offspring treated with 50 µg/mL TBTC was significantly decreased (data not provided). Comparatively, bacterial binding was increased in macrophages isolated from offspring treated with 50 µg/mL TBTC. Decreased phagocytosis (LOAEL = 15 µg/mL) and killing activities (15 µg/mL) only were observed in neutrophils. No effect on IL-1β, IL-6, or TNF-α production was noted from macrophages or neutrophils. MCP-1 production was significantly increased in neutrophils isolated from offspring treated with 50 µg/mL TBTC (Kimura et al. 2005).
Rat and mouse spleen cells were treated with TBTC for 24 hours and then LPS or PHA for 48 hours. Then, mitogen stimulation was assessed by 5-bromo-2'-deoxyuridine incorporation. TBTC inhibited cellular proliferation for both species; the inhibitory response was more potent in mice cells vs. rat cells (IC50 with LPS: 0.0025 vs. 0.007 µM, IC50 with PHA: 0.002 vs. 0.007 µM). TBTC also inhibited rat spleen proliferation that was stimulated by ConA (no data provided). TBTC also inhibited anti-CD3 antibody stimulation of mouse lymphocyte proliferation (IC50 > 0.1 µM) ).

Mode of action information
In vivo effects of TBTC on the thymus of orally treated rats are proposed to be due to the metabolite dibutyltin chloride (Snoeij et al. 1988).
The role of apoptosis is not clear. In one study the authors indicated that apoptosis does not appear to be involved in inhibition of immature thymocyte proliferation, which may lead to thymus atrophy (Gennari et al. 1997). In a separate study, the authors proposed oxidative stress plays a role in TBTC-caspase-dependent apoptosis in murine thymocytes (Sharma and Kumar 2014).
In vitro studies suggest that TBTC promotes Th2 polarization via depletion of glutathione in antigen-presenting cells, which leads to modulation of IL-10 and IL-12 production (Kato et al. 2006).

Human Data
Data from epidemiology studies No data were located.
Urethane dose-dependently decreased the percentage of CFU-GM colonies at concentrations greater than 1000 µM (data provided in graph) ).
Cytokine release from human lymphocytes was assessed in whole blood obtained from four donors. IFN-γ was evaluated in blood from three donors after PHA stimulation for 72 hours. Comparatively, TNF-α was evaluated in blood from four donors after LPS stimulation for 72 hours. IFN-γ was modulated (i.e., either increase or decrease release) in a single tested sample. TNF-α was not modulated any of the tested samples ).

Mode of action information
No data were located.

Rodent Data
Data from in vivo immunotoxicology or toxicology studies Inbred A/J mice were administered urethane (1 mg/g) via intraperitoneal injection. A biphasic response on splenic NK cell activity was noted. At one day after the injection activity was decreased 60%, activity then increased (decreased 35%), and then remained decreased until 14 days after exposure (decreased 98%). Spleen size was initially reduced, but then increased to control levels. Mitogen response (against YAC-1 or RL♂1 target cells) was initially depressed after urethane exposure and then returned to control levels (Gorelik and Herberman 1981a).
Inbred A/J mice (5-24 days old) were administered urethane (0.5 mg/g or 1 mg/g) up to 24 days old. In all tested groups, splenic NK activity was inhibited without effects on cellularity on spleens. Decreased NK activity remained until at least 8-10 weeks of age (Gorelik and Herberman 1981a).
Inbred A/J, CBA/J, and C57BL/6 mice were administered urethane (1 mg/g) via intraperitoneal injection. One day after injection, cytotoxicity (against YAC-1 target cells) of A/J and CBA/] spleen cells was significantly decreased (63% and 25%, respectively). Activity was similar to control levels at day 4. Activity then decreased in splenic cells from A/J mice (58%), while a Urethane (10%) did not deplete ear epidermis Ia-positive LCs after male BALB/c mice were treated with topical application. Similarly, urethane did not alter the density of β-glucuronidasepositive LC in C57BL mouse tails topically treated for 1 or 3 weeks (Halliday et al. 1988).
Urethane administration to pregnant ICR mice (1.5 mg/g subcutaneous injection on GD 10) produced a transient decrease in dam thymocyte cell count. At 3 days after treatment, a significant decrease in cell count was noted. By 5 days after treatment, the cell count had recovered to control levels (data in graph). A similar phenomenon was noted with thymocyte phenotypes; decrease in CD4+8+ thymocytes (88%) at day 3 after treatment was recovered by day 5. Transient changes in dam splenocyte cell count and splenocyte phenotype CD4+8-, CD4-8+, and CD4-8-also were reported. Gene expression analyses identified changes in spleen gene expression due to urethane exposure with or without immune stimulation (FCA). Increased expression of TGFβ3 was observed in the presence or absence of immune stimulation one day after treatment. IGF-I, IGF-II and IL-2 were also differentially expressed (Sharova et al. 2002).

In vitro data with cells or cell lines
Rat and mouse spleen cells were treated with urethane for 24 hours and then LPS or PHA for 48 hours. Then, mitogen stimulation was assessed by 5-bromo-2'-deoxyuridine incorporation. Urethane did not inhibit cellular proliferation in either species (data not provided). Urethane also did not modulate rat spleen proliferation that was stimulated by ConA or inhibit anti-CD3 antibody stimulation of mouse lymphocyte proliferation (data not provided) ).

Mode of action information
In vitro and in vivo studies suggest that urethane metabolism by cytochrome P450 is needed to produce the observed immunomodulatory effects (Cha et al. 2000). Macrophage effects are based on urethane effects on the inductive phase of immune responses (Foris et al. 1983).

In vitro data with cells or cell lines
Vanadium pentoxide (25 -400 µM) inhibited cell proliferation and induced cell apoptosis in a dose and time-related manner in the IL-2-independent human NK cell line, NK-92MI. Cell proliferation was maximally inhibited (78%) at 400 µM vanadium pentoxide, and the percentage of cells undergoing apoptosis increased at 12 and 24 hours of exposure (51.2 and 64.7%, respectively) as the concentration of vanadium pentoxide increased. IL-2, IL-10 and IFNγ secretion were all inhibited by vanadium pentoxide after 24 hours at the highest concentration tested. IL-2 secretion also was inhibited after 12 hours. Expression of CD25 significantly increased above background starting at 50 µM, reaching a maximal migration inhibitory factor (MIF) of 47.4% at 400 µM. A similar pattern was observed for IL-15Rα, with a maximal MIF of 55.2% at 400 μM. Fas expression began to increase at 100 µM and reached a maximal MIF of 48.9% at 400 µM, while FasL peaked at 200 µM (62.1%). Jak3 phosphorylation was increased at 12 and 24 hours after treatment with 200 and 400 µM vanadium pentoxide (data in figure), and intracellular staining showed a strong presence of pJak3 in the internal cell membranes after treatment. (Gallardo-Vera et al. 2016).

Mode of action information
Vanadium in the +2, +3, and +4 (but not the +5) valence states interacted with human FMLPactivated neutrophils and statistically significantly increased the formation of hydroxyl radicals, with additional augmentation observed in the presence of sodium azide (values not provided) (Fickl et al. 2006).
Vanadium pentoxide induced toxic effects on the IL-2-independent human NK cell line, NK-92MI, through dysregulation of signaling pathways mediated by IL-2 via increased PTEN and decreased SHP1 expression (Gallardo-Vera et al. 2018).

Data from in vivo immunotoxicology or toxicology studies
Male F344 rats were exposed to vanadium pentoxide (100 µg V/m 3 ) via inhalation, 5 hours per day for 5 days. The animals were infected with Listeria following the 5-day exposure and the bacterial burden assessed at 24, 48 and 72 hours, post-infection. Vanadium pentoxide did not have any significant effect on Listeria burdens at any of the timepoints observed (Cohen et al. 2007).
Male CD-1 mice were exposed to vanadium pentoxide (0 or an average of 1436 µg/m 3 ) via inhalation, 1 hour per day, 2 times per week over 12 weeks. An increase in the number (3.8±0.12 spleen was observed in vanadium pentoxide exposed mice, as compared to controls. These same types of changes were also observed in the bone marrow (values not provided). No statistical difference was observed in spleen weight between treated and control mice (Fortoul et al. 2008).
When male and female CD-1 mice were exposed to vanadium pentoxide (0 or 1.4 mg/m 3 ) using the same protocol as in Fortoul et al., 2008, a sex difference was observed in the expression of Ki-67, a specific proliferation marker for lymphocytes. The percentage of Ki-67 immunopositive lymphocytes increased in male mice (38.86, 41.75 and 41.91%) after 4, 8 and 12 weeks of exposure, respectively, with both cytoplasmic and nuclear expression of Ki-67 observed. In female mice, the percentage of proliferating lymphocytes increased only after the first week of exposure (34.87%) and the signal was observed only in the nucleus. Subsequent exposures did not produce significant changes in the percentage of proliferating cells in females. The authors concluded there is a role for sex hormones in potential protection against vanadium immunoxicity (Rodriguez-Lara et al. 2016).
Male CD-1 mice were exposed to vanadium pentoxide (0 or an average of 1.4 mg/m 3 ) via inhalation, 1 hour per day, 2 times per week over 12 weeks. Spleen weight of vanadium exposed animals peaked at 9 weeks (546 ± 45 vs. 274 ± 27 mg in controls) and progressively decreased afterwards (321 ± 39 mg at 12 weeks vs. 298 ± 35 mg in controls). The spleens of vanadium exposed animals had histological changes that included increased numbers of lymphocytes and megakaryocytes as compared to controls. The number of CD19 + cells was also increased within the hyperplastic germinal node (values not provided) and the mean hepatitis B surface antigen levels in immunized control mice was greater than in the exposed hosts (OD=0.39 ± 0.03 vs. 0.11 ± 0.05). The authors concluded that vanadium pentoxide induces functional changes in the spleen which appear to result in effects on the humoral immune response ).
Male CD-1 mice were exposed to vanadium pentoxide (0 or an average of 1.4 mg/m 3 ) via inhalation, 1 hour per day, 2 times per week over 4 weeks. The expression of CD11c in the thymic medulla was decreased in vanadium pentoxide exposed mice, as compared to controls (values not provided), based on immunohistochemistry. Flow cytometry also demonstrated a decrease in CD11c + and MHC-II + cells in vanadium pentoxide exposed mice, as compared to controls (values not provided). The decrease was both, in terms of number and in mean fluorescence intensity values (Ustarroz-Cano et al. 2012).
Male F344/N rats and female B6C3F1 mice were exposed to 0, 4,8, or 16 mg/m 3 vanadium pentoxide, via inhalation, 6 hours per day, 5 days per week for 16 days. Pulmonary inflammation was assessed via analysis of BAL fluid. Significant alterations in the percentage of recoverable macrophages and neutrophils (NOAEL 4 mg/m 3 ), and increased lung protein and lysozyme in male rats (LOAEL 4 mg/m 3 ) were observed. In female mice, an increase in lymphocytes, protein and lysozymes was observed (LOAEL 4 mg/m 3 ). No effects were observed on systemic immunity as evidenced by a normal response to Klebsiella pneumoniae (National Toxicology Program 2002).
The induction of pulmonary inflammation was examined in three different strains of mice [A/J (sensitive strain for pulmonary inflammation and carcinogenesis), BALB/c (intermediate sensitivity), and C57Bl/6J (resistant)]. Mice were aspirated with vanadium pentoxide (4 mg/kg) or phosphate-buffered saline, four times per week, with BALF collected at 6 hours, and 1, 3, 6 and 21 days. In A/J mice, vanadium pentoxide increased BALF levels of total cells (95.7%) inflammatory markers (PMNs,macrophages and lymphocytes,74.6,99.5,and 623.8%,respectively). Levels of inflammatory chemokines (keratinocyte-derived chemokine, macrophage inflammatory protein-2 and monocyte chemoattractant protein 1), transcription factor activity (NFκB and c-Fos) and signaling pathway activation (MAPK) were increased with highest levels observed in A/J mice followed by BALB/c and then C57BL/6J mice (data in graphs). All results returned to baseline 21 days post exposure (Rondini et al. 2010).

In vitro data with cells or cell lines
No data were located.

Mode of action information
Rondini and colleagues (2010) reported that vanadium pentoxide impacts pulmonary levels of inflammatory markers, induction of chemokines, and modulation of transcription factors Alterations in macrophage mediated functions have been associated with vanadium exposure (Cohen et al. 1996).
The ability of several vanadium compounds to increase mRNA levels of cytokines in BALF was investigated in female CD rats. Rats received 42 or 420 µg of vanadium pentoxide or phosphatebuffered saline by intratracheal instillation. BALF was collected at times ranging from 1 hour to 10 days. Influx of neutrophils was significantly increased 24 hours after exposure to vanadium pentoxide and peaked 24-48 hours post exposure (data in graph). Macrophage inflammatory protein-2 mRNA expression levels were significantly elevated in vanadium pentoxide treated rats at 1 to 48-hour timepoints, as compared to controls (Pierce et al. 1996).