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National Toxicology Program. NTP Technical Report on the Toxicity Studies of Abrasive Blasting Agents Administered by Inhalation to F344/NTac Rats and Sprague Dawley (Hsd:Sprague Dawley® SD®) Rats: Toxicity Report 91 [Internet]. Research Triangle Park (NC): National Toxicology Program; 2020 Jun.
NTP Technical Report on the Toxicity Studies of Abrasive Blasting Agents Administered by Inhalation to F344/NTac Rats and Sprague Dawley (Hsd:Sprague Dawley® SD®) Rats: Toxicity Report 91 [Internet].
Show detailsF.1. Methods
Studies evaluating the potential immunotoxicity of blasting sand or specular hematite in core study male and special study female Sprague Dawley rats exposed by inhalation for up to 39 weeks were conducted at Battelle Toxicology Northwest (Richmond, WA). Animals were exposed by whole-body inhalation to blasting sand or specular hematite at concentrations of 0, 15, 30, or 60 mg/m3, 5 days per week for up to 39 weeks. For each compound, groups of eight unimmunized male and eight immunized and eight unimmunized female rats per time point were selected for immunotoxicological evaluations.
Bronchoalveolar lavage (BAL) fluid and blood samples for analyses of antinuclear antibodies were collected from unimmunized male and unimmunized female rats. On the day of study termination (1 day after the last exposure) rats were euthanized by intraperitoneal injection of pentobarbital. The spleens of immunized and unimmunized female rats were aseptically removed from the animals, placed in tubes containing Earle’s Balanced Salt Solution (EBSS) with 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), supplemented with gentamicin (a bacteriostat), and a “wet” weight was obtained. Tubes containing the whole spleens were placed on crushed ice and shipped overnight to the designated immunotoxicology contract laboratory, Virginia Commonwealth University (VCU) in Richmond, VA. Lavage samples were centrifuged, and the supernatant (i.e., BAL fluid) was removed and frozen at −70°C. Serum was prepared from the whole blood samples and frozen at −70°C. The BAL fluid and serum samples were shipped to VCU on dry ice and stored frozen at −70°C for analysis.
Spleens from immunized and unimmunized female rats were processed for immunotoxicological evaluation. Assessment of immune function in lymphoid tissues up to 24 hours after tissue collection has been shown to produce comparable results to studies conducted on freshly harvested tissues.76 Single-cell suspensions were prepared by mashing spleens as previously described.77 Spleen cell suspensions from sheep red blood cell (sRBC)-immunized animals were centrifuged and resuspended in 6 mL of EBSS with 15 mM HEPES. Cell suspensions for the unimmunized animals were centrifuged and resuspended in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS).
The primary IgM response to sRBCs was enumerated using a modification of the hemolytic plaque assay as described by White et al.78 Rats were immunized with 2 × 108 sRBCs by intravenous injection 4 days before study termination. Single-cell suspensions were prepared from the spleens of immunized rats and resuspended in 6 mL of EBSS. An aliquot of cells was added to the test tube containing guinea pig complement, sRBCs, and warm agar. After thoroughly mixing, the test tube mixture was plated in a petri dish, covered with a microscope slide cover slip, and incubated at 37°C for 3 hours. Cell counts were performed on the 6 mL cell suspensions, and the numbers of cells/spleen, antibody-forming cells (AFCs) /106 spleen cells, and AFCs/spleen were determined.
An enzyme-linked immunosorbent assay (ELISA) system, developed at VCU,79 was used to determine serum titers of antigen-specific IgM in blood obtained from the same animals immunized with sRBCs for the AFC assay. One day before the ELISA was conducted, sRBC membrane high-salt release antigens (1 mg/mL) were diluted 1:100 in phosphate buffered saline (PBS) and applied to Immulon® 2 microtiter plates (Thermo-Fisher Scientific, Inc., Waltham, MA) (100 μL/well) and incubated at 4oC overnight. Before each subsequent step, plates were washed three times with 200 μL per well per wash of PBS with 0.05% Tween® 20 (Thermo-Fisher Scientific, Inc., Waltham, MA) (assay buffer). After the plates were incubated for 60 minutes with assay buffer (175 μL/well), serum samples diluted with assay buffer were added to wells of the appropriate plates for a final volume of 100 μL of diluted serum. After 60 minutes of incubation at room temperature, the plates were washed, and the secondary antibody [affinity-purified horseradish peroxidase-conjugated goat anti-rat IgM antibody (Southern BioTech, Birmingham, AL) diluted 1:500 in assay buffer] was added (100 μL/well) and allowed to incubate for approximately 60 minutes. Plates were subsequently washed, and peroxidase substrate [2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid), Sigma-Aldrich, St. Louis, MO] was added (100 μL/well). The color in each well was read at 405 nm on a Molecular Devices (San Jose, CA) plate reader after a 45-minute incubation period. Results were obtained using SoftMax® (v. 2.32, Molecular Devices). The antibody titer was defined to be the reciprocal of the dilution at which the sample absorbance had an optical density (OD) of 0.5 and was obtained by interpolating within the linear portion of the log-log regression curve. If a sample did not reach an OD of 0.5, it was assigned a titer of the starting dilution.
For immunophenotyping studies, single-cell suspensions were prepared from the spleens of unimmunized female rats and resuspended in 6 mL of RPMI 1640 media supplemented with 10% FBS. Red blood cells were removed via ammonium chloride lysis and 1.0 × 106 spleen cells per well were seeded into a 96-well microtiter plate. Individual wells contained 100 μL of spleen cells and 100 μL of a surface marker specific monoclonal antibody conjugated to fluorescein isothiocyanate (FITC) or phycoerythrin (PE). The specific monoclonal antibodies used were OX19 conjugated to PE to enumerate T cells (CD5+), OX38 conjugated to FITC to enumerate CD4+ cells, and OX8 conjugated to FITC to enumerate CD8+ cells. For both the CD4+ and CD8+ cells, a double label with OX19 and OX38 or OX8 was used. OX33 conjugated to FITC was used to enumerate CD45RA+ B cells. Splenic natural killer (NK) cells were enumerated using a FITC-conjugated NKR-P1A antibody and OX8 conjugated to PE, and splenic macrophages were enumerated using HIS36 antibody conjugated to PE. The final dilution of each antibody in the well was 1:80.
An appropriate isotype control was run for each of the antibodies used. Following the initial staining with antibody and washing with staining buffer, 100 μL of propidium iodide (PI) was added to each well to determine viability. Following 5 minutes of incubation with PI, the cells were washed once with staining buffer and then enumerated on a Becton Dickinson FACScan™ flow cytometer (Beckton, Dickinson and Company San Jose, CA). Five thousand cells were counted for each sample.
T cell proliferation was measured in splenocytes from unimmunized female rats to assess cell-mediated immunity. Flat-bottom 96-well microtiter plates were coated with either rat anti-CD3 monoclonal antibody (diluted to 1 μg/mL in PBS; 0.1 mL/well) or PBS only (100 μL/well) and incubated overnight. The plates were then washed with 200 μL of sterile PBS before the addition of spleen cells. Single-cell suspensions were prepared under aseptic conditions, centrifuged, and resuspended in 6 mL of RPMI 1640 media supplemented with 10% FBS at a concentration of 5 × 106 cells/mL. One hundred μL of spleen cells and 100 μL of medium (RPMI 1640 supplemented with 10% FBS and 50 μM 2-mercaptoethanol) were added to each well. The splenocytes were cultured in both uncoated and anti-CD3 coated wells in quadruplicate for 3 days. Eighteen to 24 hours before harvest on day 3, 1 μCi of 3H-thymidine was added to each well. Cells were harvested onto Wallac filtermats (Perkin Elmer, Waltham, MA) using a Harvester 96 Mach IIIM cell harvester (Tomtec, Hamden, CT) and counted using a 1450 Microbeta® Trilux Liquid Scintillation and Luminescence Counter (Perkin Elmer). The incorporation of 3H-thymidine into DNA was used as a measure of cell proliferation, and the data were expressed as counts per minute (CPM)/5 × 105 cells.
NK cell activity was evaluated in spleen cells from unimmunized female rats as a measure of innate immune function. Spleen cells were prepared under aseptic conditions, resuspended in 6 mL of RPMI 1640 medium supplemented with 10% FBS, and adjusted to six concentrations, 2 × 107, 107, 5 × 106, 2.5 × 106, 1.25 × 106 and 0.625 × 106 cells/mL. YAC-1 murine lymphoma cells, maintained in a stock culture at VCU, were used as the target cells in this assay. Cultured YAC-1 cells were harvested, centrifuged, and resuspended in RPMI 1640 medium supplemented with 10% FBS at a concentration of 107 cells/mL. YAC-1 cells were radiolabeled by incubation with 200 μCi of 51Cr for 90 minutes at 37°C. Following the incubation, the cells were washed three times in RPMI 1640 with 10% FBS, counted, and adjusted to 105 nucleated cells/mL. The target cells were added to each well in a volume of 0.1 mL (105 YAC-1 cells/mL). Spleen cells were added in a volume of 0.1 mL to each of two replicate wells of target cells at each effector concentration to obtain effector-to-target (E:T) ratios of 200:1, 100:1, 50:1, 25:1, 12.5:1, and 6.25:1. Maximum release of 51Cr was determined by adding 0.1 mL of the labeled YAC-1 cells and 0.1 mL 0.1% Triton X-100 to each of 12 replicate wells. Spontaneous release was determined by adding 0.1 mL of medium to each of 12 replicate cultures containing the targets. The plates were incubated for 4 hours at 37°C under 5% CO2. Following the incubation, the plates were centrifuged at 250 × g for 10 minutes, and 0.1 mL of the supernatant was removed from each well and counted in a Wallac 1480 Wizard® 3” gamma counter Perkin Elmer. The mean percent cytotoxicity at each effector concentration was determined for each exposed group and compared to the values for the chamber control group. The results are expressed as percentage of cytotoxicity as follows:
% Cytotoxicity = (CPMexp − CPMspon)/(CPMtotal − CPMspon) × 100,
where CPMexp = counts per minute in experimental wells, CPMspon = spontaneous release, and CPMtotal = total release upon addition of 0.1% Triton X-100.
BAL fluid received at VCU was stored frozen at −70°C until analysis. Samples were thawed overnight in a refrigerator at 2° to 4° C. Cytokine levels were determined on a single 25 μL aliquot of each sample of BAL fluid using FlowCytomix™ bead array kits (eBioscience, Inc., San Diego, CA) according to the manufacturer’s instructions. Cytokines evaluated were: interleukin (IL)-1α, monocyte chemotactic protein (MCP)-1, tumor necrosis factor (TNF)-α, granulocyte-macrophage colony stimulating factor (GM-CSF), IL-4, and interferon (IFN)-γ. Cytokine concentrations were determined by interpolation against standard curves generated for each cytokine using standards included in the FlowCytomix™ bead array kits. A value of 0 was assigned to serum samples that had levels below the limit of detection for any given cytokine.
An ELISA was used to analyze serum for autoantibodies against nuclear antigens. The ELISA was conducted using a modification of commercially available human antinuclear antibody (ANA) ELISA kits (INOVA Diagnostics, Inc., San Diego, CA). Modifications included the use of affinity-purified horseradish peroxidase-conjugated goat anti-rat heavy- and light-chain specific immunoglobulin [Ig(H+L)] (Southern BioTech), diluted 1:1000 in PBS as the secondary antibody, and 3,3′,5,5′-tetramethylbenzidine (BD Biosciences, San Jose, CA) as the substrate. ANA-positive serum (for use as a positive control on each plate) was generated in female Brown Norway rats following exposure to 1 mg/kg mercuric chloride by subcutaneous injection three times per week for 2 weeks. According to the manufacturer, the antigens bound to the surface of the wells of the precoated plates included chromatin (dsDNA and histones), Sm/RNP, SS-A, SS-B, Scl-70, centromere, PCNA, Jo-1, mitochondria (M-2), ribosomal-P protein, and highly purified extracts from HEp-2 nuclei and nucleoli.
Serum samples were diluted 1:50 in sample diluent and added in a volume of 100 μL per well to the precoated plates. Following 30 minutes of incubation at room temperature, the plates were washed three times, and secondary antibody was added to all wells in a volume of 100 μL/well. After 30 minutes, the plates were washed four times, and 100 μL of substrate was added to each well. Five minutes later, stop solution (2 N sulfuric acid) was added in a volume of 100 μL/well. The OD of each well was read at 450 nm on a Molecular Devices plate reader within 30 minutes of the addition of the stop solution. Results were obtained using SoftMax v. 2.32. The mean OD of the background wells (wells containing no sample) was subtracted from the OD obtained for each sample and positive control to obtain corrected OD values. For each time point (4-, 16-, 26-, and 39-week samples), the mean and standard deviation (SD) of the corrected OD values for the chamber control animals was determined. The data were then analyzed to determine whether any sample fell outside of the 99% confidence interval of the mean for the chamber control group. Per the manufacturer’s instructions, if a sample had a corrected OD greater than the mean + 3 × the SD, the sample was classified as “positive,” indicating that the sample had a significantly greater level of ANAs than that of the chamber control group. Samples within the limits of the 99% confidence interval were classified as “negative,” which indicated that the ANA response of that sample was not significantly different from that of the appropriate chamber control group. Positive control serum was run on each plate to demonstrate the assay was capable of detecting serum ANAs. Results are presented as incidence of ANA-positive samples and as the percentage of positive responders.
F.2. Results
F.2.1. Blasting Sand
The results of the assay measuring the IgM AFC response to sRBCs (T-dependent antigen) using spleen cells from immunized female rats are shown in Table F-1 and Table F-2 at weeks 5 and 27, respectively. No effects were observed on total spleen cell numbers or on the AFC response of female rats following exposure to blasting sand at either time point. Similarly, blasting sand exposure did not result in any differences in serum anti-sRBC IgM antibody titers at either week 5 or week 27 (Table F-3 and Table F-4).
Spleen weights were unaffected in immunized female rats that were exposed to blasting sand for 27 weeks (Table F-2) and unimmunized female rats exposed to blasting sand for 26 weeks (Table F-12). Total spleen cell numbers in unimmunized females were unaffected following 4 weeks of blasting sand exposure (Table F-5). However, after 26 weeks, total spleen cell numbers were significantly lower (27%) in the 60 mg/m3 group of unimmunized females (Table F-7).
Phenotypic analysis was conducted to evaluate multiple spleen cell populations, including: B cells (OX33+), T cells (CD5+), TH cells (CD4+CD5+), TCTL cells (CD8+CD5+), NK cells (NK+CD8+), and macrophages (His36+). In unimmunized female rats exposed to blasting sand for 4 weeks, the absolute numbers of B cells, T cells, TCTL cells, NK cells, and macrophages were all unaffected, although a positive trend occurred in absolute T cell numbers (Table F-5). TH cells were increased 19% and 37% at the 15 and 60 mg/m3 exposure levels, respectively. When evaluated as percent values (Table F-6), significant increases occurred in T cells (22%) and TH cells (32%) at the 30 mg/m3 exposure level. In addition, TH cells were significantly increased in the 60 mg/m3 group. The percentages of B cells, TCTL cells, NK cells, and macrophages were unaffected, although a negative trend occurred in the percentage of B cells.
Following exposure of unimmunized female rats to blasting sand for 26 weeks, absolute numbers of B cells were lower, in an exposure concentration-related manner, at all exposure levels (Table F-7). In addition, absolute NK cell and macrophage numbers were significantly lower in the 60 mg/m3 group. Negative trends occurred in all cell populations with the exception of TH cells, when evaluated as absolute values. When evaluated in terms of percent values at both 30 and 60 mg/m3, B cells were significantly decreased and TH cells were significantly increased (Table F-8). No other significant differences were observed.
As a measure of the effects of blasting sand exposure on cell-mediated immunity, the anti-CD3 antibody-mediated proliferative response was evaluated in unimmunized female rats. After 4 weeks of exposure, no significant effects were observed on either unstimulated or anti-CD3-stimulated proliferation (Table F-9). In contrast, the anti-CD3-stimulated proliferative response of spleen cells from female rats exposed to 60 mg/m3 blasting sand for 26 weeks was significantly increased (68%; Table F-10).
NK cell activity was assessed in unimmunized female rats using six Effector:Target (E:T) ratios in a 4-hour 51Cr-release assay, and the results were expressed in terms of percent cytotoxicity. No significant differences occurred NK cell activity between the exposed and chamber control groups at any E:T ratio following exposure to blasting sand for either 4 weeks (Table F-11) or 26 weeks (Table F-12).
Cytokine levels in the BAL fluid of unimmunized male and female rats exposed to chamber control air or blasting sand were evaluated in a single 25 μL sample using a commercially available cytometric bead array kit, as described in the methods section. The six cytokines evaluated were: IL-1α, MCP-1, TNF-α, IFN-γ, GM-CSF, and IL-4. IFN-γ was not detected in any of the samples at any time point (Table F-13 and Table F-14). After 4 weeks of exposure to blasting sand, levels of IL-1α, TNF-α, and IL-4 were significantly increased in the 15 mg/m3 group of female rats (Table F-13). MCP-1 and GM-CSF levels were not affected at this time point (Table F-3 and Table F-14). In female rats exposed to blasting sand for 26 weeks, MCP-1 levels were significantly increased in an exposure concentration-dependent manner. MCP-1 was not detected in BAL fluid from any of the chamber control animals, therefore percent increases for this cytokine could not be calculated. GM-CSF and IL-4 levels were each significantly decreased in the 30 and 60 mg/m3 groups, whereas TNF-α was significantly decreased only in the 60 mg/m3 group. IL-1 levels were unaffected. In male rats exposed to 60 mg/m3 blasting sand for 4 weeks, MCP-1 levels were significantly increased (441%) compared to the chamber control group (Table F-14). No other significant differences between the exposed and chamber control groups occurred at this time point. MCP-1 levels were significantly increased in all groups of male rats exposed to blasting sand at all of the later time points evaluated (16, 26, and 39 weeks; Table F-14 and Figure F-1).
Following exposure to chamber control air or blasting sand for 4 or 26 weeks, serum from unimmunized female rats was analyzed for the presence of autoantibodies. After 4 weeks of exposure, serum ANA levels of one female rat in the 15 mg/m3 group and three female rats in the 60 mg/m3 group were greater than the upper limit of the 99% confidence interval of the chamber control group mean (Table F-15). No female rats in the 30 mg/m3 group had ANA levels exceeding that upper limit. Statistical analysis of these incidences using the Fisher exact chi-square test indicated that none of the groups exposed to blasting sand were significantly different from the chamber control group at this time point. At the 26-week time point, only one female rat in the 15 mg/m3 group had serum ANA levels outside the 99% confidence interval of the chamber control group mean. No other animals exceeded the upper confidence limit. The Fisher exact chi-square test indicated no significant differences in incidence between groups exposed to blasting sand and the chamber control group at this time point. Serum from unimmunized male rats exposed to chamber control air or blasting sand for 4, 16, 26, or 39 weeks was also analyzed for autoantibody levels. After 26 weeks of exposure, one, two, and three male rats in the 15, 30, and 60 mg/m3 groups, respectively, had serum ANA levels outside the upper limit of the 99% confidence interval of the chamber control group mean (Table F-15). Statistical analysis of these incidences using the Fisher exact chi-square test indicated that none of the male groups exposed to blasting sand were significantly different from the chamber control group at this time point. No other male rats in any of the exposed groups at any other time point had serum ANA levels outside the 99% confidence intervals of the chamber control group means.
F.2.2. Specular Hematite
The results of the assay measuring the IgM AFC response to sRBCs (T-dependent antigen) using spleen cells from immunized female rats are shown in Table F-16 and Table F-17 at weeks 5 and 27, respectively. No effects were observed on spleen weights, total spleen cell numbers, or the AFC response in immunized female rats following exposure to specular hematite at either time point. Exposure to specular hematite also did not affect the serum anti-sRBC IgM antibody titers at either week 5 (Table F-18) or week 27 (Table F-19).
The effects of specular hematite exposure on spleen weights in female rats that were not immunized with sRBCs are shown in Table F-26 and Table F-27; no significant differences were observed between the specular hematite-exposed groups and the chamber control groups at either weeks 4 or 26.
Phenotypic analysis of spleen cells from unimmunized female rats was conducted for multiple cell populations, including: B cells (OX33+), T cells (CD5+), TH cells (CD4+CD5+), TCTL cells (CD8+CD5+), NK cells (NK+CD8+), and macrophages (His36+). Total spleen cell numbers were unaffected by specular hematite exposure for 4 or 26 weeks, as shown in Table F-20 and Table F-22, respectively. Following exposure to specular hematite for 4 weeks, no effects were observed on the absolute values of the various spleen cell phenotypes (Table F-20). When evaluated as percent values, a significant decrease (11%) in the percentage of B cells occurred in female rats exposed to 15 mg/m3 specular hematite for 4 weeks (Table F-21); no effects on percent values of any other cell population were observed at week 4.
No effects were observed on the absolute numbers of B cells, T cells, TH cells, TCTL cells, NK cells, or macrophages in unimmunized female rats exposed to specular hematite for 26 weeks (Table F-22). When evaluated as percent values, B cells, TH cells, NK cells, and macrophages were all unaffected (Table F-23). Significant decreases of 20% and 29% occurred in the percentages of T cells and TCTL cells in female rats exposed to 30 mg/m3 specular hematite, and significant negative trends were observed for each of these cell populations. However, no significant changes were observed at the higher exposure concentration of 60 mg/m3.
The anti-CD3 antibody-stimulated proliferative response was evaluated in unimmunized female rats following 4 and 26 weeks of exposure to specular hematite. At 4 weeks, significant increases in the basal (unstimulated) proliferative response occurred in rats exposed to 15 or 30 mg/m3 specular hematite (Table F-24). No effects were observed on the unstimulated response in the 60 mg/m3 group. No significant effects were observed on anti-CD3-stimulated proliferation in any of the groups exposed to specular hematite for 4 weeks. Exposure to specular hematite for 26 weeks did not affect basal or anti-CD3-stimulated proliferation in cells obtained from the spleens of exposed rats (Table F-25).
Following exposure to specular hematite for 4 weeks, NK cell activity in the spleen of unimmunized female rats was unaffected at all E:T ratios examined (Table F-26). In unimmunized female rats exposed to specular hematite for 26 weeks, significant increases in NK cell activity occurred at the 50:1 and the 25:1 E:T ratios for the 30 and 60 mg/m3 exposure groups, respectively (Table F-27). No other significant effects were observed, although positive trends occurred at all E:T ratios in rats exposed to specular hematite for 26 weeks.
Two of eight unimmunized female rats exposed to 60 mg/m3 specular hematite for 4 weeks had detectable levels of IFN-γ in their BAL fluid (Table F-28). However, there were no statistically significant changes in any of the six cytokines measured in female rats exposed for 4 weeks. After 26 weeks of exposure to specular hematite, MCP-1 levels were significantly increased in BAL fluid from 30 and 60 mg/m3 female rats. In the BAL fluid of unimmunized male rats exposed to specular hematite for 4 weeks, no significant changes occurred in any of the six cytokines measured (Table F-29). However, exposure concentration-related increases in MCP-1 levels did occur following 16 weeks of exposure, and the increases were significant in the 30 and 60 mg/m3 groups (Table F-29 and Figure F-2). Exposure to specular hematite for 26 weeks also resulted in exposure concentration-dependent increases in MCP-1 levels in male rats, and the increases were statistically significant at all exposure levels. Exposure to specular hematite for 39 weeks resulted in significant increases in MCP-1 levels in BAL fluid of male rats exposed to 30 or 60 mg/m3. Levels of all other cytokines evaluated in male rats were not detectable or were not significantly different from those in the chamber control animals at the 16-, 26-, or 39-week time points (Table F-29).
After 4 weeks of exposure to specular hematite, one unimmunized 15 mg/m3 female rat had a serum ANA level outside the upper limit of the 99% confidence interval for the chamber control group mean (Table F-30). No other female rats in any exposed group had serum ANA levels outside the upper limit of the 99% confidence interval of the chamber control group mean after 4 weeks of exposure. In unimmunized female rats exposed to specular hematite for 26 weeks, two animals in the 30 mg/m3 group had serum ANA levels greater than the 99% confidence interval upper limit for the chamber control group. One 15 mg/m3 and one 60 mg/m3 unimmunized male rat exposed to specular hematite for 4 and 39 weeks, respectively, had serum ANA levels outside the 99% confidence interval upper limits for the chamber control groups. The Fisher exact chi-square test indicated that none of the incidences of ANA-positive responses in groups exposed to specular hematite were significantly different from those in the chamber control groups at any of the time points examined for male or female rats.
F.3. Conclusions
Exposure to blasting sand by inhalation for up to 27 weeks produced minimal immunotoxic effects in female Sprague Dawley rats. The functional responses of humoral and innate immunity were unaffected. Total spleen cell numbers were lower, whereas anti-CD3-mediated proliferation was increased in female rats exposed to 60 mg/m3 blasting sand for 26 weeks. Levels of MCP-1 were increased in an exposure concentration-dependent manner in the BAL fluid of both male and female Sprague Dawley rats exposed to blasting sand, beginning at week 4 in males and at week 26 in females. Antinuclear autoantibody levels in serum from male and female Sprague Dawley rats were not affected by exposure to blasting sand.
Similarly, exposure to specular hematite by inhalation for up to 27 weeks in female Sprague Dawley rats and up to 39 weeks in male Sprague Dawley rats produced minimal effects on the immune measures examined. The functional responses of humoral, cell-mediated and innate immunity were generally unaffected in female Sprague Dawley rats exposed to specular hematite. MCP-1 was the only cytokine in the BAL fluid that was modulated by exposure to specular hematite. Levels of MCP-1 were significantly increased in rats exposed to specular hematite, beginning at week 16 in males and at week 26 in females. Exposure to specular hematite did not significantly affect serum levels of antinuclear autoantibodies in male or female Sprague Dawley rats.
Table F-1Spleen IgM Antibody-Forming Cell Response to the T-Dependent Antigen Sheep Erythrocytes in Female Sprague Dawley Rats Exposed to Blasting Sand for Five Weeks (Day 4 Response)
| Total Spleen Cells (×107) | IgM AFC/106 Spleen Cells | IgM AFC/Spleen (×103) | ||
|---|---|---|---|---|
| n | 8 | 8 | 8 | |
| Chamber Control | 88.42 ± 6.79 | 623 ± 116 | 532 ± 103 | |
| Blasting Sand | ||||
| 15 mg/m3 | 87.65 ± 4.10 | 960 ± 260 | 875 ± 250 | |
| 30 mg/m3 | 93.95 ± 4.99 | 811 ± 149 | 762 ± 148 | |
| 60 mg/m3 | 91.55 ± 3.28 | 1,033 ± 211 | 969 ± 222 | |
| H/NH | H | H | H | |
| Trend Analysis | NS | NS | NS | |
Female Sprague Dawley rats were exposed to chamber control air or blasting sand by inhalation. Four days before euthanasia, the rats were immunized with 2 × 108 sheep red blood cells (sRBCs). On the day of euthanasia, spleens were placed into tubes containing medium and sent to Virginia Commonwealth University (Richmond, VA) on ice for next-day cell preparation. Spleens were prepared into single-cell suspensions and the number of IgM sRBC antibody-forming cells (AFCs) was determined. Values represent the mean ± standard error derived from the number of animals indicated by n.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance; differences from the chamber control group are not significant by Dunnett’s t-test. Jonckheere’s test was used to assess the significance of exposure concentration-related trends.
NS = not significant.
Table F-2Spleen IgM Antibody-Forming Cell Response to the T-Dependent Antigen Sheep Erythrocytes in Female Sprague Dawley Rats Exposed to Blasting Sand for 27 Weeks (Day 4 Response)
| Spleen Weight (mg) | Total Spleen Cells (×107) | IgM AFC/106 Spleen Cells | IgM AFC/Spleen (×103) | |
|---|---|---|---|---|
| n | 8 | 8 | 8 | 8 |
| Chamber Control | 758 ± 45 | 87.50 ± 5.08 | 1,093 ± 403 | 1,006 ± 404 |
| Blasting Sand | ||||
| 15 mg/m3 | 729 ± 45 | 76.44 ± 4.14 | 1,056 ± 417 | 832 ± 346 |
| 30 mg/m3 | 789 ± 54 | 78.32 ± 4.28 | 1,555 ± 571 | 1,212 ± 449 |
| 60 mg/m3 | 790 ± 82 | 78.06 ± 5.14 | 688 ± 178 | 578 ± 195 |
| H/NH | H | H | H | H |
| Trend Analysis | NS | NS | NS | NS |
Female Sprague Dawley rats were exposed to chamber control air or blasting sand by inhalation. Four days before euthanasia, the rats were immunized with 2 × 108 sheep red blood cells (sRBCs). On the day of euthanasia, spleens were placed into tubes containing medium and sent to Virginia Commonwealth University (Richmond, VA) on ice for next-day cell preparation. Spleens were prepared into single-cell suspensions and the number of IgM sRBC antibody-forming cells (AFCs) was determined. Values represent the mean ± standard error derived from the number of animals indicated by n.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance; differences from the chamber control group are not significant by Dunnett’s t-test. Jonckheere’s test was used to assess the significance of exposure concentration-related trends.
NS = not significant.
Table F-3Serum IgM Antibody Titers to the T-Dependent Antigen Sheep Erythrocytes in Female Sprague Dawley Rats Exposed to Blasting Sand for Five Weeks
| Number of Animals | Serum Titer Log2 | |
|---|---|---|
| Chamber Control | 8 | 6.168 ± 0.317 |
| Blasting Sand | ||
| 15 mg/m3 | 8 | 6.609 ± 0.388 |
| 30 mg/m3 | 8 | 6.272 ± 0.618 |
| 60 mg/m3 | 8 | 6.343 ± 0.529 |
| H/NH | – | H |
| Trend Analysis | – | NS |
Female Sprague Dawley rats were exposed to chamber control air or blasting sand by inhalation. On the day of euthanasia, serum was obtained via cardiac puncture and frozen. Serum samples were shipped to Virginia Commonwealth University (Richmond, VA) on dry ice for analysis. Values represent the mean ± standard error derived from the indicated number of animals.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance; differences from the chamber control group are not significant by Dunnett’s t-test. Jonckheere’s test was used to assess the significance of exposure concentration-related trends.
NS = not significant.
Table F-4Serum IgM Antibody Titers to the T-Dependent Antigen Sheep Erythrocytes in Female Sprague Dawley Rats Exposed to Blasting Sand for 27 Weeks
| Number of Animals | Serum Titer Log2 | |
|---|---|---|
| Chamber Control | 8 | 7.544 ± 0.529 |
| Blasting Sand | ||
| 15 mg/m3 | 8 | 7.524 ± 0.817 |
| 30 mg/m3 | 8 | 6.298 ± 0.435 |
| 60 mg/m3 | 8 | 6.659 ± 0.625 |
| H/HN | – | H |
| Trend Analysis | – | NS |
Female Sprague Dawley rats were exposed to chamber control air or blasting sand by inhalation. On the day of euthanasia, serum was obtained via cardiac puncture and frozen. Serum samples were shipped to Virginia Commonwealth University (Richmond, VA) on dry ice for analysis. Values represent the mean ± standard error derived from the indicated number of animals.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance; differences from the chamber control group are not significant by Dunnett’s t-test. Jonckheere’s test was used to assess the significance of exposure concentration-related trends.
NS = not significant.
Table F-5Splenocyte Surface Marker Differential in Female Sprague Dawley Rats Exposed to Blasting Sand for Four Weeks (Absolute Values)
| Total Spleen Cells (×107) | OX33+a | CD5+b | CD4+CD5+c | CD8+CD5+d | NK+CD8+e | HIS36+f | |
|---|---|---|---|---|---|---|---|
| n | 8 | 8 | 8 | 8 | 8 | 8 | 8 |
| Chamber Control | 79.56 ± 1.25 | 450.7 ± 8.6 | 161.1 ± 7.3 | 83.8 ± 3.9 | 69.5 ± 3.8 | 29.1 ± 1.8 | 34.2 ± 3.6 |
| Blasting Sand | |||||||
| 15 mg/m3 | 79.09 ± 3.45 | 449.9 ± 22.6 | 179.0 ± 9.5 | 99.7 ± 7.0* | 76.9 ± 5.0 | 24.2 ± 1.8 | 33.2 ± 2.9 |
| 30 mg/m3 | 76.41 ± 4.55 | 435.8 ± 29.5 | 186.9 ± 10.5 | 106.2 ± 9.1 | 71.6 ± 5.7 | 26.9 ± 2.5 | 37.0 ± 4.8 |
| 60 mg/m3 | 80.70 ± 4.08 | 432.8 ± 26.4 | 193.1 ± 14.9 | 115.0 ± 13.9* | 76.7 ± 4.4 | 27.5 ± 2.6g | 37.7 ± 3.8 |
| H/NH | NH | NH | H | NH | H | H | H |
| Trend Analysis | NS | NS | p ≤ 0.05 | p ≤ 0.01 | NS | NS | NS |
- *
Significantly different (p ≤ 0.05) from the chamber control group by Wilcoxon’s rank test.
Female Sprague Dawley rats were exposed to chamber control air or blasting sand by inhalation. On the day of euthanasia, spleens were placed into tubes containing medium and sent to Virginia Commonwealth University (Richmond, VA) on ice for next-day cell preparation. B cells, T cells, T-subsets, NK cells, and macrophages were enumerated. Surface marker values are expressed as the absolute number per spleen × 106 and represented as the mean ± standard error derived from the number of animals indicated by n.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance; differences from the chamber control group are not significant by Dunnett’s t-test. Nonhomogeneous data were evaluated using a nonparametric analysis of variance. When significant differences occurred, exposed groups were compared to the chamber control group using Wilcoxon’s rank test. Jonckheere’s test was used to assess the significance of exposure concentration-related trends.
NS = not significant.
- a
B cell.
- b
T cell.
- c
Helper/DTH − T cell.
- d
Cytotoxic T cell.
- e
Natural killer cell.
- f
Macrophage.
- g
n = 7; no sample aliquot was available to analyze the NK cell marker for one animal due to a technical error.
Table F-6Splenocyte Surface Marker Differential in Female Sprague Dawley Rats Exposed to Blasting Sand for Four Weeks (Percent Values)
| OX33+a | CD5+b | CD4+CD5+c | CD8+CD5+d | NK+CD8+e | HIS36+f | |
|---|---|---|---|---|---|---|
| n | 8 | 8 | 8 | 8 | 8 | 8 |
| Chamber Control | 56.7 ± 1.2 | 20.3 ± 0.9 | 10.5 ± 0.5 | 8.7 ± 0.5 | 3.7 ± 0.2 | 4.3 ± 0.4 |
| Blasting Sand | ||||||
| 15 mg/m3 | 56.8 ± 0.8 | 22.8 ± 1.1 | 12.6 ± 0.7 | 9.8 ± 0.7 | 3.1 ± 0.2 | 4.2 ± 0.4 |
| 30 mg/m3 | 56.9 ± 1.3 | 24.8 ± 1.3* | 13.9 ± 0.9* | 9.6 ± 0.8 | 3.5 ± 0.2 | 4.9 ± 0.7 |
| 60 mg/m3 | 53.6 ± 1.2 | 23.8 ± 1.0 | 14.0 ± 1.0* | 9.6 ± 0.5 | 3.4 ± 0.3g | 4.7 ± 0.4 |
| H/NH | H | H | H | H | H | H |
| Trend Analysis | p ≤ 0.05 | p ≤ 0.01 | p ≤ 0.01 | NS | NS | NS |
- *
Significantly different (p ≤ 0.05) from the chamber control group by Dunnett’s t-test.
Female Sprague Dawley rats were exposed to chamber control air or blasting sand by inhalation. On the day of euthanasia, spleens were placed into tubes containing medium and sent to Virginia Commonwealth University (Richmond, VA) on ice for next-day cell preparation. B cells, T cells, T-subsets, NK cells, and macrophages were enumerated. Values are expressed as percent of total spleen cells counted for each animal (Table F-5) and represented as the mean ± standard error derived from the number of animals indicated by n.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance. When significant differences occurred, exposed groups were compared to the chamber control group using Dunnett’s t-test. Jonckheere’s test was used to assess the significance of exposure concentration-related trends.
NS = not significant.
- a
B cell.
- b
T cell.
- c
Helper/DTH − T cell.
- d
Cytotoxic T cell.
- e
Natural killer cell.
- f
Macrophage.
- g
n = 7; there was no sample aliquot available to analyze the NK marker for one animal due to a technical error.
Table F-7Splenocyte Surface Marker Differential in Female Sprague Dawley Rats Exposed to Blasting Sand for 26 Weeks (Absolute Values)
| Total Spleen Cells (×107) | OX33+a | CD5+b | CD4+CD5+c | CD8+CD5+d | NK+CD8+e | HIS36+f | |
|---|---|---|---|---|---|---|---|
| n | 8 | 8 | 8 | 8 | 8 | 8 | 8 |
| Chamber Control | 76.88 ± 3.64 | 434.2 ± 24.8 | 148.6 ± 12.1 | 73.6 ± 5.4 | 53.4 ± 5.4 | 26.6 ± 1.9 | 46.1 ± 6.0 |
| Blasting Sand | |||||||
| 15 mg/m3 | 64.32 ± 4.74 | 345.7 ± 26.2* | 123.6 ± 12.3 | 63.9 ± 5.4 | 50.8 ± 6.7 | 22.9 ± 2.4 | 47.3 ± 6.2 |
| 30 mg/m3 | 63.31 ± 4.01 | 318.3 ± 21.8** | 129.8 ± 10.3 | 79.7 ± 7.3 | 44.0 ± 3.4 | 21.3 ± 1.8 | 41.3 ± 5.2 |
| 60 mg/m3 | 55.79 ± 3.08** | 275.5 ± 15.3** | 111.1 ± 3.4 | 65.5 ± 3.6 | 37.8 ± 2.1 | 16.1 ± 1.7** | 24.8 ± 2.0* |
| H/NH | H | H | NH | H | NH | H | H |
| Trend Analysis | p ≤ 0.01 | p ≤ 0.01 | p ≤ 0.05 | NS | p ≤ 0.01 | p ≤ 0.01 | p ≤ 0.01 |
- *
Significantly different (p ≤ 0.05) from the chamber control group by Dunnett’s t-test.
- **
p ≤ 0.01.
Female Sprague Dawley rats were exposed to chamber control air or blasting sand by inhalation. On the day of euthanasia, spleens were placed into tubes containing medium and sent to Virginia Commonwealth University (Richmond, VA) on ice for next-day cell preparation. B cells, T cells, T-subsets, NK cells, and macrophages were enumerated. Surface marker values are expressed as the absolute number per spleen × 106 and represented as the mean ± standard error derived from the number of animals indicated by n.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance. When significant differences occurred, exposed groups were compared to the chamber control group using Dunnett’s t-test. Nonhomogeneous data were evaluated using a nonparametric analysis of variance; differences from the chamber control group are not significant by Wilcoxon’s rank test. Jonckheere’s test was used to assess the significance of exposure concentration-related trends.
NS = not significant.
- a
B cell.
- b
T cell.
- c
Helper/DTH − T cell.
- d
Cytotoxic T cell.
- e
Natural killer cell.
- f
Macrophage.
Table F-8Splenocyte Surface Marker Differential in Female Sprague Dawley Rats Exposed to Blasting Sand for 26 Weeks (Percent Values)
| OX33+a | CD5+b | CD4+CD5+c | CD8+CD5+d | NK+CD8+e | HIS36+f | |
|---|---|---|---|---|---|---|
| n | 8 | 8 | 8 | 8 | 8 | 8 |
| Chamber Control | 56.4 ± 1.4 | 19.3 ± 1.3 | 9.6 ± 0.5 | 7.0 ± 0.7 | 3.5 ± 0.2 | 6.1 ± 0.8 |
| Blasting Sand | ||||||
| 15 mg/m3 | 53.7 ± 1.0 | 19.1 ± 1.0 | 9.9 ± 0.4 | 7.8 ± 0.8 | 3.6 ± 0.4 | 7.4 ± 0.9 |
| 30 mg/m3 | 50.3 ± 1.4** | 20.6 ± 1.1 | 12.5 ± 0.7** | 7.1 ± 0.6 | 3.3 ± 0.1 | 6.7 ± 0.9 |
| 60 mg/m3 | 49.4 ± 0.7** | 20.2 ± 0.9 | 11.9 ± 0.7* | 6.9 ± 0.4 | 2.9 ± 0.2 | 4.5 ± 0.5 |
| H/NH | H | H | H | H | NH | H |
| Trend Analysis | p ≤ 0.01 | NS | p ≤ 0.01 | NS | p ≤ 0.05 | NS |
- *
Significantly different (p ≤ 0.05) from the chamber control group by Dunnett’s t-test.
- **
p ≤ 0.01.
Female Sprague Dawley rats were exposed to chamber control air or blasting sand by inhalation. On the day of euthanasia, spleens were placed into tubes containing medium and sent to Virginia Commonwealth University (Richmond, VA) on ice for next-day cell preparation. B cells, T cells, T-subsets, NK cells, and macrophages were enumerated. Values are expressed as percent of total spleen cells counted for each animal (Table F-7) and represented as the mean ± standard error derived from the number of animals indicated by n.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance. When significant differences occurred, exposed groups were compared to the chamber control group using Dunnett’s t-test. Nonhomogeneous data were evaluated using a nonparametric analysis of variance; differences from the chamber control group are not significant by Wilcoxon’s rank test. Jonckheere’s test was used to assess the significance of exposure concentration-related trends.
NS = not significant.
- a
B cell.
- b
T cell.
- c
Helper/DTH − T cell.
- d
Cytotoxic T cell.
- e
Natural killer cell.
- f
Macrophage.
Table F-9Spleen Cell Proliferative Response to Anti-CD3 Stimulation in Female Sprague Dawley Rats Exposed to Blasting Sand for Four Weeks
| Chamber Control | 15 mg/m3 | 30 mg/m3 | 60 mg/m3 | H/NH | Trend Analysis | |
|---|---|---|---|---|---|---|
| n | 8 | 8 | 8 | 8 | – | – |
| Spleen Cell Cultures | ||||||
| Unstimulated | 7,252 ± 870 | 6,958 ± 853 | 7,097 ± 1,099 | 12,667 ± 3,384 | NH | NS |
| Stimulated | 74,323 ± 9,547 | 78,623 ± 8,681 | 82,460 ± 13,382 | 80,696 ± 6,730 | H | NS |
| Total Spleen Cells × 107 | 79.56 ± 1.25 | 79.09 ± 3.45 | 76.41 ± 4.55 | 80.70 ± 4.08 | NH | NS |
Female Sprague Dawley rats were exposed to chamber control air or blasting sand by inhalation. On the day of euthanasia, spleens were placed into tubes containing medium and sent to Virginia Commonwealth University (Richmond, VA) on ice for next-day cell preparation. Spleens were prepared into single-cell suspensions and incubated in flat-bottom microtiter plates. The medium for the proliferative assay was RPMI supplemented with 10% FBS and 50 μM 2-mercaptoethanol. The spleen cells were cultured in either nontreated or treated anti-CD3 wells. Prior to harvest, the cells were pulsed with 3H-thymidine for 18 to 24 hours. The cultured cell data are presented as mean ± standard error derived from four replicate cultures from the number of animals indicated by n.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance. Nonhomogeneous data were evaluated using a nonparametric analysis of variance. Differences from the chamber control group are not significant by Dunnett’s t-test (homogenous data) or Wilcoxon’s rank test (nonhomogeneous data). Jonckheere’s test was used to assess the significance of exposure concentration-related trends.
NS = not significant.
Table F-10Spleen Cell Proliferative Response to Anti-CD3 Stimulation in Female Sprague Dawley Rats Exposed to Blasting Sand for 26 Weeks
| Chamber Control | 15 mg/m3 | 30 mg/m3 | 60 mg/m3 | H/NH | Trend Analysis | |
|---|---|---|---|---|---|---|
| n | 8 | 8 | 8 | 8 | – | |
| Spleen Cell Cultures | ||||||
| Unstimulated | 8,536 ± 868 | 8,701 ± 999 | 8,819 ± 1,529 | 8,934 ± 521 | H | NS |
| Stimulated | 167,122 ± 21,667 | 195,523 ± 27,959 | 243,652 ± 31,761 | 281,322 ± 32,010* | H | p ≤ 0.01 |
| Total Spleen Cells × 107 | 76.88 ± 3.64 | 64.32 ± 4.74 | 63.31 ± 4.01 | 55.79 ± 3.08** | H | p ≤ 0.01 |
- *
Significantly different (p ≤ 0.05) from the chamber control group by the Dunnett test.
- **
p ≤ 0.01.
Female Sprague Dawley rats were exposed to chamber control air or blasting sand by inhalation. On the day of euthanasia, spleens were placed into tubes containing medium and sent to Virginia Commonwealth University (Richmond, VA) on ice for next-day cell preparation. Spleens were prepared into single-cell suspensions and incubated in flat-bottom microtiter plates. The medium for the proliferative assay was RPMI supplemented with 10% FBS and 50 μM 2-mercaptoethanol. The spleen cells were cultured in either nontreated or treated anti-CD3 wells. Prior to harvest, the cells were pulsed with 3H-thymidine for 18 to 24 hours. The cultured cell data are presented as mean ± standard error CPM cells derived from four replicate cultures from the number of animals indicated by n.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance. When significant differences occurred, exposed groups were compared to the chamber control group using Dunnett’s t-test. Jonckheere’s test was used to assess the significance of exposure concentration-related trends.
NS = not significant.
Table F-11Natural Killer Cell Activity in Female Sprague Dawley Rats Exposed to Blasting Sand for Four Weeks
| Chamber Control | 15 mg/m3 | 30 mg/m3 | 60 mg/m3 | H/NH | Trend Analysis | |
|---|---|---|---|---|---|---|
| n | 8 | 8 | 8 | 8 | – | – |
| Effector:Target Ratio | ||||||
| 200:1 | 43.3 ± 3.1 | 34.9 ± 3.6 | 35.3 ± 5.3 | 40.4 ± 3.1 | H | NS |
| 100:1 | 27.3 ± 2.6 | 20.7 ± 2.6 | 22.8 ± 3.4 | 24.4 ± 2.4 | H | NS |
| 50:1 | 15.1 ± 1.4 | 12.0 ± 1.7 | 12.5 ± 2.1 | 13.8 ± 1.3 | H | NS |
| 25:1 | 9.4 ± 1.3 | 7.2 ± 1.2 | 7.9 ± 1.5 | 7.8 ± 0.8 | H | NS |
| 12.5:1 | 6.2 ± 0.8 | 4.9 ± 0.9 | 5.1 ± 0.9 | 6.4 ± 0.9 | H | NS |
| 6.25:1 | 4.3 ± 0.5 | 3.5 ± 0.6 | 3.7 ± 0.6 | 4.2 ± 0.5 | H | NS |
Female Sprague Dawley rats were exposed to chamber control air or blasting sand by inhalation. On the day of euthanasia, spleens were placed into tubes containing medium and sent to Virginia Commonwealth University (Richmond, VA) on ice for next-day cell preparation. Spleens were prepared into single-cell suspensions and were assayed 4 hours later for natural killer cell activity using 51Cr-labeled YAC-1 cells as the target. Spontaneous release over the 4-hour incubation period was 9.6% of maximum release. NK cell activity values represent the mean ± standard error percent cytotoxicity derived from the number of animals indicated by n.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance; differences from the chamber control group are not significant by Dunnett’s t-test. Jonckheere’s test was used to assess the significance of exposure concentration-related trends.
NS = not significant.
Table F-12Natural Killer Cell Activity in Female Sprague Dawley Rats Exposed to Blasting Sand for 26 Weeks
| Chamber Control | 15 mg/m3 | 30 mg/m3 | 60 mg/m3 | H/NH | Trend Analysis | |
|---|---|---|---|---|---|---|
| n | 8 | 8 | 8 | 8 | – | – |
| Spleen Wt. (mg) | 714 ± 41 | 725 ± 60 | 817 ± 67 | 739 ± 33 | H | NS |
| Effector:Target Ratio | ||||||
| 200:1 | 31.8 ± 2.0 | 30.6 ± 2.0 | 39.2 ± 1.5 | 35.2 ± 3.0 | H | p ≤ 0.05 |
| 100:1 | 21.5 ± 1.5 | 23.0 ± 1.2 | 27.5 ± 1.9 | 25.2 ± 3.1 | H | p ≤ 0.05 |
| 50:1 | 12.9 ± 1.2 | 12.4 ± 0.7 | 16.8 ± 0.7 | 13.1 ± 1.9 | NH | NS |
| 25:1 | 7.6 ± 0.9 | 6.9 ± 0.6 | 9.3 ± 0.5 | 8.1 ± 1.3 | H | NS |
| 12.5:1 | 4.4 ± 0.7 | 4.1 ± 0.5 | 5.6 ± 0.4 | 4.6 ± 0.9 | H | NS |
| 6.25:1 | 3.1 ± 0.5 | 3.2 ± 0.3 | 3.6 ± 0.2 | 3.2 ± 0.6 | NH | NS |
Female Sprague Dawley rats were exposed to chamber control air or blasting sand by inhalation. On the day of euthanasia, spleens were placed into tubes containing medium and sent to Virginia Commonwealth University (Richmond, VA) on ice for next-day cell preparation. Spleens were prepared into single-cell suspensions and were assayed 4 hours later for natural killer cell activity using 51Cr-labeled YAC-1 cells as the target. Spontaneous release over the 4-hour incubation period was 8.2% of maximum release. NK cell activity values represent the mean ± standard error percent cytotoxicity derived from the number of animals indicated by n.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance. Nonhomogeneous data were evaluated using a nonparametric analysis of variance. Differences from the chamber control group are not significant by Dunnett’s t-test (homogenous data) or Wilcoxon’s rank test (nonhomogeneous data). Jonckheere’s test was used to assess the significance of exposure concentration-related trends.
NS = not significant.
Table F-13Bronchoalveolar Lavage Fluid Cytokine Levels in Female Sprague Dawley Rats Exposed to Blasting Sand for 4 or 26 Weeks
| Chamber Control | 15 mg/m3 | 30 mg/m3 | 60 mg/m3 | H/NH | Trend Analysis | |
|---|---|---|---|---|---|---|
| n | 8 | 8 | 8 | 8 | – | – |
| Week 4 | ||||||
| IL-1α | 17.30 ± 9.67 | 71.77 ± 15.41** | 21.07 ± 13.15 | 12.58 ± 6.16 | H | p ≤ 0.01 |
| MCP-1 | ND | ND | ND | 26.12 ± 13.49 | NH | p ≤ 0.01 |
| TNF-α | 25.08 ± 4.38 | 50.96 ± 5.64** | 20.12 ± 6.88 | 19.12 ± 4.43 | H | p ≤ 0.05 |
| IFN-γ | ND | ND | ND | ND | NA | NA |
| GM-CSF | 55.84 ± 9.84 | 85.46 ± 13.34 | 49.70 ± 12.05 | 43.48 ± 9.75 | H | NS |
| IL-4 | 2.86 ± 0.31 | 4.64 ± 0.45** | 2.71 ± 0.27 | 2.19 ± 0.34 | H | NS |
| Week 26 | ||||||
| IL-1α | 20.00 ± 11.69 | 26.83 ± 10.06 | 1.37 ± 1.37 | 9.67 ± 9.67 | NH | p ≤ 0.01 |
| MCP-1 | ND | 78.52 ± 24.33** | 336.77 ± 53.14** | 1,094.75 ± 164.11** | NH | p ≤ 0.01 |
| TNF-α | 36.60 ± 4.37 | 26.96 ± 4.59 | 24.22 ± 4.33 | 8.80 ± 6.45** | H | p ≤ 0.01 |
| IFN-γ | ND | ND | ND | ND | NA | NA |
| GM-CSF | 67.97 ± 4.40 | 64.16 ± 3.49 | 30.18 ± 11.65* | 24.50 ± 12.56** | NH | p ≤ 0.01 |
| IL-4 | 3.22 ± 0.32 | 2.83 ± 0.18 | 1.60 ± 0.50* | 0.70 ± 0.47** | H | p ≤ 0.01 |
- *
Significantly different (p ≤ 0.05) from the chamber control group by Dunnett’s test (homogeneous data) or Wilcoxon’s test (nonhomogeneous data).
- **
p ≤ 0.01.
Female Sprague Dawley rats were exposed to chamber control air or blasting sand by inhalation. On the day of euthanasia, bronchoalveolar lavage fluid was collected and frozen. Samples were sent to Virginia Commonwealth University (Richmond, VA) on dry ice for sample analysis. Cytokine levels were analyzed in 25 μL aliquots of sample using FlowCytomixTM bead array kits according to kit instructions. Values represent the mean ± standard error pg/mL of each cytokine derived from the number of animals indicated by n.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance. When significant differences occurred, exposed groups were compared to the chamber control group using Dunnett’s t-test. Nonhomogeneous data were evaluated using a nonparametric analysis of variance. When significant differences occurred, exposed groups were compared to the chamber control group using Wilcoxon’s rank test. Jonckheere’s test was used to assess the significance of concentration-related trends.
NS = not significant; ND = not detected; NA = not applicable.
Table F-14Bronchoalveolar Lavage Fluid Cytokine Levels in Male Sprague Dawley Rats Exposed to Blasting Sand for 4, 16, 26, or 39 Weeks
| Chamber Control | 15 mg/m3 | 30 mg/m3 | 60 mg/m3 | H/NH | Trend Analysis | |
|---|---|---|---|---|---|---|
| n | 8 | 8 | 8 | 8 | – | – |
| Week 4 | ||||||
| IL-α | 11.95 ± 5.90 | 9.82 ± 6.13 | 15.30 ± 8.19 | 13.20 ± 6.97 | H | NS |
| MCP-1 | 8.15 ± 1.72 | 6.75 ± 1.48 | 9.07 ± 2.11 | 44.07 ± 5.83** | NH | p ≤ 0.01 |
| TNF-α | 8.75 ± 4.38 | 7.82 ± 3.47 | 5.18 ± 3.71 | 2.32 ± 2.19 | H | p ≤ 0.01 |
| IFN-γ | ND | ND | ND | ND | NA | NA |
| GM-CSF | 15.80 ± 7.45 | 16.70 ± 7.44 | 24.99 ± 11.01 | 11.71 ± 8.29 | H | p ≤ 0.05 |
| IL-4 | 0.09 ± 0.09 | 0.03 ± 0.03 | 0.19 ± 0.15 | 0.06 ± 0.06 | NH | p ≤ 0.01 |
| Week 16 | ||||||
| IL-1 | 5.20 ± 5.20 | 2.38 ± 1.70 | 6.65 ± 5.12 | 0.73 ± 0.73 | NH | p ≤ 0.01 |
| MCP-1α | 5.67 ± 2.13 | 19.67 ± 1.28** | 83.96 ± 10.48** | 290.69 ± 39.56** | NH | p ≤ 0.01 |
| TNF-α | 3.84 ± 3.84 | ND | 0.12 ± 0.12 | ND | NH | p ≤ 0.01 |
| IFN-γ | ND | ND | ND | ND | NA | NA |
| GM-CSF | 6.25 ± 6.25 | 5.80 ± 5.80 | 1.76 ± 1.76 | ND | NH | p ≤ 0.01 |
| IL-4 | 0.06 ± 0.06 | ND | ND | ND | NH | p ≤ 0.01 |
| Week 26 | ||||||
| IL-1 | 8.05 ± 4.48 | 2.79 ± 2.51 | 7.05 ± 4.32 | 18.10 ± 9.11 | NH | NS |
| MCP-1α | 8.52 ± 1.28 | 46.50 ± 7.15** | 202.67 ± 40.54** | 513.65 ± 58.86** | NH | p ≤ 0.01 |
| TNF-α | 2.99 ± 2.15 | 2.34 ± 1.80 | 22.70 ± 5.10 | 14.76 ± 5.74 | NH | NS |
| IFN-γ | ND | ND | ND | ND | NA | NA |
| GM-CSF | 5.75 ± 3.23 | 5.02 ± 4.28 | 46.16 ± 10.47 | 47.40 ± 10.76 | NH | NS |
| IL-4 | ND | ND | 2.28 ± 0.37 | 2.04 ± 0.49 | NH | NS |
| Week 39 | ||||||
| IL-1α | ND | ND | ND | ND | NA | NA |
| MCP-1 | 2.25 ± 1.47 | 55.40 ± 22.76** | 219.68 ± 63.29** | 872.04 ± 108.00** | NH | p ≤ 0.01 |
| TNF-α | ND | ND | ND | ND | NA | NA |
| IFN-γ | ND | ND | ND | ND | NA | NA |
| GM-CSF | ND | ND | ND | ND | NA | NA |
| IL-4 | ND | ND | ND | ND | NA | NA |
- **
Significantly different (p ≤ 0.01) from the chamber control group by Wilcoxon’s rank test.
Male Sprague Dawley rats were exposed to chamber control air or blasting sand by inhalation. On the day of euthanasia, bronchoalveolar lavage fluid was collected and frozen. Samples were sent to Virginia Commonwealth University (Richmond, VA) on dry ice for sample analysis. Cytokine levels were analyzed in 25 μL aliquots of sample using FlowCytomixTM bead array kits according to kit instructions. Values represent the mean ± standard error pg/mL for each cytokine derived from the number of animals indicated by n.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance; differences from the chamber control group are not significant by Dunnett’s t-test. Nonhomogeneous data were evaluated using a nonparametric analysis of variance. When significant differences occurred, exposed groups were compared to the chamber control group using Wilcoxon’s rank test. Jonckheere’s test was used to assess the significance of concentration-related trends.
NS = not significant; ND = not detected; NA = not applicable.
Table F-15Antinuclear Antibody-Positive Responses in Sprague Dawley Rats Exposed to Blasting Sand
| Chamber Control | 15 mg/m3 | 30 mg/m3 | 60 mg/m3 | |
|---|---|---|---|---|
| n | 8 | 8 | 8 | 8 |
| Male | ||||
| Week 4 | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| Week 16 | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| Week 26 | 0 (0) | 1 (13) | 2 (25) | 3 (38) |
| Week 39 | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| Female | ||||
| Week 4 | 0 (0) | 1 (13) | 0 (0) | 3 (38) |
| Week 26 | 0 (0) | 1 (13) | 0 (0) | 0 (0) |
Male and female Sprague Dawley rats were exposed to chamber control air or blasting sand by inhalation. On the day of euthanasia, serum was collected and frozen. Samples were sent to Virginia Commonwealth University (Richmond, VA) on dry ice for sample analysis. Samples were diluted 1:50 and analyzed for the presence of antinuclear antibodies by enzyme-linked immunosorbent assay. Results are presented as the incidence (percent) of positive responses. A positive response was defined as a sample having an optical density that exceeded the upper limit of the 99% confidence interval of the mean of the chamber control group at the same time point. Differences in incidence from the chamber control group are not significant by the Fisher exact chi-square test.
Table F-16Spleen IgM Antibody-Forming Cell Response to the T-Dependent Antigen Sheep Erythrocytes in Female Sprague Dawley Rats Exposed to Specular Hematite for Five Weeks (Day 4 Response)
| Spleen Weight (mg) | Total Spleen Cells (×107) | IgM AFC/106Spleen Cells | IgM AFC/Spleen(×103) | |
|---|---|---|---|---|
| n | 8 | 8 | 8 | 8 |
| Chamber Control | 539 ± 30 | 65.09 ± 4.82 | 642 ± 162 | 431 ± 109 |
| Specular Hematite | ||||
| 15 mg/m3 | 502 ± 35 | 67.27 ± 4.08 | 676 ± 147 | 468 ± 113 |
| 30 mg/m3 | 592 ± 40 | 76.05 ± 6.08 | 858 ± 173 | 659 ± 149 |
| 60 mg/m3 | 597 ± 27 | 69.02 ± 3.76 | 920 ± 171 | 650 ± 137 |
| H/NH | H | H | H | H |
| Trend Analysis | NS | NS | NS | NS |
Female Sprague Dawley rats were exposed to chamber control air or specular hematite by inhalation. Four days before euthanasia, the rats were immunized with 2 × 108 sheep red blood cells (sRBCs). On the day of euthanasia, spleens were placed in tubes containing medium and sent to Virginia Commonwealth University (Richmond, VA) on ice for next-day cell preparation. Spleens were prepared into single-cell suspensions and the number of IgM sRBC antibody-forming cells (AFCs) was determined. Values represent the mean ± standard error derived from the number of animals indicated by n.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance; differences from the chamber control group are not significant by Dunnett’s t-test. Jonckheere’s test was used to assess the significance of exposure concentration-related trends.
NS = not significant.
Table F-17Spleen IgM Antibody-Forming Cell Response to the T-Dependent Antigen Sheep Erythrocytes in Female Sprague Dawley Rats Exposed to Specular Hematite for 27 Weeks (Day 4 Response)
| Spleen Weight (mg) | Total Spleen Cells (× 107) | IgM AFC/106 Spleen Cells | IgM AFC/Spleen (× 103) | |
|---|---|---|---|---|
| n | 8 | 8 | 8 | 8 |
| Chamber Control | 620 ± 25a | 79.88 ± 3.58a | 525 ± 108 | 419 ± 87a |
| Specular Hematite | ||||
| 15 mg/m3 | 629 ± 36 | 85.22 ± 4.60 | 520 ± 136 | 450 ± 131 |
| 30 mg/m3 | 599 ± 13 | 70.81 ± 2.90 | 669 ± 113 | 465 ± 73 |
| 60 mg/m3 | 634 ± 25 | 71.63 ± 3.07 | 822 ± 207 | 610 ± 160 |
| H/NH | H | H | H | H |
| Trend Analysis | NS | p ≤ 0.05 | NS | NS |
Female Sprague Dawley rats were exposed to chamber control air or specular hematite by inhalation. Four days before euthanasia, the rats were immunized with 2 × 108 sheep red blood cells (sRBCs). On the day of euthanasia, spleens were placed in tubes containing medium and sent to Virginia Commonwealth University (Richmond, VA) on ice for next-day cell preparation. Spleens were prepared into single-cell suspensions and the number of IgM sRBC antibody-forming cells (AFCs) was determined. Values represent the mean ± standard error derived from the number of animals indicated by n.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance; differences from the chamber control group are not significant by Dunnett’s t-test. Jonckheere’s test was used to assess the significance of exposure concentration-related trends.
NS = not significant.
- a
n = 7.
Table F-18Serum IgM Antibody Titers to the T-Dependent Antigen Sheep Erythrocytes in Female Sprague Dawley Rats Exposed to Specular Hematite for Five Weeks
| Number of Animals | Serum Titer Log2 | |
|---|---|---|
| Control | 8 | 5.939 ± 0.208 |
| Specular Hematite | ||
| 15 mg/m3 | 8 | 5.370 ± 0.273 |
| 30 mg/m3 | 8 | 6.335 ± 0.275 |
| 60 mg/m3 | 8 | 6.353 ± 0.252 |
| H/NH | – | H |
| Trend Analysis | – | NS |
Female Sprague Dawley rats were exposed to chamber control air or specular hematite by inhalation. On the day of euthanasia, serum was obtained via cardiac puncture and frozen. Serum samples were shipped to Virginia Commonwealth University (Richmond, VA) on dry ice for analysis. Values represent the mean ± standard error derived from the indicated number of animals.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance; differences from the chamber control group are not significant by Dunnett’s t-test. Jonckheere’s test was used to assess the significance of exposure concentration-related trends.
NS = not significant.
Table F-19Serum IgM Antibody Titers to the T-Dependent Antigen Sheep Erythrocytes in Female Sprague Dawley Rats Exposed to Specular Hematite for 27 Weeks
| Number of Animals | Serum Titer Log2 | |
|---|---|---|
| Chamber Control | 8 | 6.844 ± 0.505 |
| Specular Hematite | ||
| 15 mg/m3 | 8 | 5.756 ± 0.577 |
| 30 mg/m3 | 8 | 6.058 ± 0.567 |
| 60 mg/m3 | 8 | 6.375 ± 0.322 |
| H/NH | – | H |
| Trend Analysis | – | NS |
Female Sprague Dawley rats were exposed to chamber control air or specular hematite by inhalation. On the day of euthanasia, serum was obtained via cardiac puncture and frozen. Serum samples were shipped to Virginia Commonwealth University (Richmond, VA) on dry ice for analysis. Values represent the mean ± standard error derived from the indicated number of animals.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance; differences from the chamber control group are not significant by Dunnett’s t-test. Jonckheere’s test was used to assess the significance of exposure concentration-related trends.
NS = not significant.
Table F-20Splenocyte Surface Marker Differential in Female Sprague Dawley Rats Exposed to Specular Hematite for Four Weeks (Absolute Values)
| Total Spleen Cells (×107) | OX33+a | CD5+b | CD4+CD5+c | CD8+CD5+d | NK+CD8+e | HIS36+f | |
|---|---|---|---|---|---|---|---|
| n | 8 | 8 | 8 | 8 | 8 | 8 | 8 |
| Chamber Control | 59.79 ± 3.79 | 346.3 ± 23.2 | 133.1 ± 10.8 | 76.7 ± 8.1 | 49.1 ± 3.9 | 24.1 ± 2.3 | 25.0 ± 3.1 |
| Specular Hematite | |||||||
| 15 mg/m3 | 60.50 ± 4.02 | 309.6 ± 20.6 | 158.5 ± 16.9 | 82.5 ± 7.1 | 62.8 ± 6.5 | 20.4 ± 1.8 | 29.5 ± 3.0 |
| 30 mg/m3 | 60.71 ± 3.52 | 352.9 ± 25.9 | 130.0 ± 8.8 | 71.3 ± 6.4 | 52.4 ± 3.6 | 22.6 ± 1.9 | 33.6 ± 4.1 |
| 60 mg/m3 | 61.34 ± 3.44 | 341.4 ± 26.2 | 137.2 ± 9.1 | 72.6 ± 5.5 | 55.4 ± 3.5 | 22.8 ± 1.5 | 25.5 ± 3.0 |
| H/NH | H | H | H | H | H | H | H |
| Trend Analysis | NS | NS | NS | NS | NS | NS | NS |
Female Sprague Dawley rats were exposed to chamber control air or specular hematite by inhalation. On the day of euthanasia, spleens were placed into tubes containing medium and sent to Virginia Commonwealth University (Richmond, VA) on ice for next-day cell preparation. B cells, T cells, T-subsets, NK cells, and macrophages were enumerated. Surface marker values are expressed as the absolute number per spleen × 106 and represented as the mean ± standard error derived from the number of animals indicated by n.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance; differences from the chamber control group are not significant by Dunnett’s t-test. Jonckheere’s test was used to assess the significance of exposure concentration-related trends.
NS = not significant.
- a
B cell.
- b
T cell.
- c
Helper/DTH − T cell.
- d
Cytotoxic T cell.
- e
Natural killer cell.
- f
Macrophage.
Table F-21Splenocyte Surface Marker Differential in Female Sprague Dawley Rats Exposed to Specular Hematite for Four Weeks (Percent Values)
| OX33+a | CD5+b | CD4+CD5+c | CD8+CD5+d | NK+CD8+e | HIS36+f | ||
|---|---|---|---|---|---|---|---|
| n | 8 | 8 | 8 | 8 | 8 | 8 | |
| Chamber Control | 58.0 ± 1.4 | 22.3 ± 1.4 | 12.9 ± 1.3 | 8.2 ± 0.4 | 4.0 ± 0.3 | 4.2 ± 0.4 | |
| Specular Hematite | |||||||
| 15 mg/m3 | 51.4 ± 1.5** | 26.1 ± 1.5 | 13.7 ± 0.6 | 10.3 ± 0.7 | 3.4 ± 0.3 | 4.9 ± 0.5 | |
| 30 mg/m3 | 57.8 ± 1.3 | 21.7 ± 1.3 | 11.9 ± 0.9 | 8.7 ± 0.5 | 3.7 ± 0.2 | 5.5 ± 0.5 | |
| 60 mg/m3 | 55.3 ± 1.4 | 22.5 ± 1.2 | 11.9 ± 0.8 | 9.1 ± 0.5 | 3.9 ± 0.5 | 4.1 ± 0.3 | |
| H/NH | H | H | H | H | H | H | |
| Trend Analysis | NS | NS | NS | NS | NS | NS | |
- **
Significantly different (p ≤ 0.01) from the chamber control group by Dunnett’s t-test.
Female Sprague Dawley rats were exposed to chamber control air or specular hematite by inhalation. On the day of euthanasia, spleens were placed into tubes containing medium and sent to Virginia Commonwealth University (Richmond, VA) on ice for next-day cell preparation. B cells, T cells, T-subsets, NK cells, and macrophages were enumerated. Values are expressed as percent of total spleen cells counted for each animal (Table F-20) and represented as the mean ± standard error derived from the number of animals indicated by n.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance. When significant differences occurred, exposed groups were compared to the chamber control group using Dunnett’s t-test. Jonckheere’s test was used to assess the significance of exposure concentration-related trends.
NS = not significant.
- a
B cell.
- b
T cell.
- c
Helper/DTH − T cell.
- d
Cytotoxic T cell.
- e
Natural killer cell.
- f
Macrophage.
Table F-22Splenocyte Surface Marker Differential in Female Sprague Dawley Rats Exposed to Specular Hematitefor 26 Weeks (Absolute Values)
| Total Spleen Cells (×107) | OX33+a | CD5+b | CD4+CD5+c | CD8+CD5+d | NK+CD8+e | HIS36+f | |
|---|---|---|---|---|---|---|---|
| n | 8 | 8 | 8 | 8 | 8 | 8 | 8 |
| Chamber Controlg | 65.63 ± 3.93 | 299.0 ± 32.6 | 206.0 ± 12.7 | 97.2 ± 9.5 | 95.3 ± 6.0 | 14.5 ± 0.8 | 30.8 ± 3.9 |
| Specular Hematite | |||||||
| 15 mg/m3 | 73.36 ± 4.02 | 330.8 ± 16.6 | 231.8 ± 14.7 | 124.1 ± 11.5 | 106.6 ± 11.3 | 18.8 ± 1.8 | 30.1 ± 2.7 |
| 30 mg/m3 | 64.82 ± 3.37 | 305.0 ± 19.1 | 164.4 ± 17.1 | 97.4 ± 11.5 | 66.0 ± 7.8 | 16.0 ± 1.2 | 31.8 ± 3.2 |
| 60 mg/m3 | 66.66 ± 2.67 | 305.6 ± 18.3 | 191.9 ± 13.4 | 105.6 ± 8.9 | 77.1 ± 8.3 | 17.9 ± 1.1 | 24.6 ± 1.8 |
| H/NH | H | H | H | H | H | H | H |
| Trend Analysis | NS | NS | p ≤ 0.05 | NS | p ≤ 0.05 | NS | NS |
Female Sprague Dawley rats were exposed to chamber control air or specular hematite by inhalation. On the day of euthanasia, spleens were placed into tubes containing medium and sent to Virginia Commonwealth University (Richmond, VA) on ice for next-day cell preparation. B cells, T cells, T-subsets, NK cells, and macrophages were enumerated. Surface marker values are expressed as the absolute number per spleen × 106 and represented as the mean ± standard error derived from the number of animals indicated by n.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance; differences from the chamber control group are not significant by Dunnett’s t-test. Jonckheere’s test was used to assess the significance of exposure concentration-related trends.
NS = not significant.
- a
B cell.
- b
T cell.
- c
Helper/DTH − T cell.
- d
Cytotoxic T cell.
- e
Natural killer cell.
- f
Macrophage.
- g
n = 7; the values for one chamber control animal were excluded due to very low cell viabilities for all markers.
Table F-23Splenocyte Surface Marker Differential in Female Sprague Dawley Rats Exposed to Specular Hematite for 26 Weeks (Percent Values)
| Exposure | OX33+a | CD5+b | CD4+CD5+c | CD8+CD5+d | NK+CD8+e | HIS36+f |
|---|---|---|---|---|---|---|
| n | 8 | 8 | 8 | 8 | 8 | 8 |
| Chamber Controlg | 44.9 ± 2.8 | 31.6 ± 1.3 | 14.6 ± 0.7 | 14.6 ± 0.6 | 2.2 ± 0.1 | 4.6 ± 0.5 |
| Specular Hematite | ||||||
| 15 mg/m3 | 45.3 ± 1.4 | 31.7 ± 1.4 | 16.8 ± 1.1 | 14.3 ± 0.9 | 2.6 ± 0.3 | 4.1 ± 0.3 |
| 30 mg/m3 | 46.9 ± 1.1 | 25.4 ± 2.2* | 14.8 ± 1.2 | 10.3 ± 1.2* | 2.5 ± 0.1 | 4.9 ± 0.3 |
| 60 mg/m3 | 45.7 ± 1.6 | 28.8 ± 1.5 | 15.8 ± 1.1 | 11.6 ± 1.0 | 2.7 ± 0.2 | 3.8 ± 0.3 |
| H/NH | H | H | H | H | NH | H |
| Trend Analysis | NS | p ≤ 0.05 | NS | p ≤ 0.01 | NS | NS |
- *
Significantly different (p ≤ 0.05) from the chamber control group by Dunnett’s test.
Female Sprague Dawley rats were exposed to chamber control air or specular hematite by inhalation. On the day of euthanasia, spleens were placed into tubes containing medium and sent to Virginia Commonwealth University (Richmond, VA) on ice for next-day cell preparation. B cells, T cells, T-subsets, NK cells, and macrophages were enumerated. Values are expressed as percent total spleen cells counted for each animal (Table F-22) and represented as the mean ± standard error derived from the number of animals indicated by n.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance. When significant differences occurred, exposed groups were compared to the chamber control group using Dunnett’s t-test. Nonhomogeneous data were evaluated using a nonparametric analysis of variance; differences from the chamber control group are not significant by Wilcoxon’s test. Jonckheere’s test was used to assess the significance of exposure concentration-related trends.
NS = not significant.
- a
B cell.
- b
T cell.
- c
Helper/DTH − T cell.
- d
Cytotoxic T cell.
- e
Natural killer cell.
- f
Macrophage.
- g
n = 7; the values for one chamber control animal were excluded due to very low cell viabilities for all markers.
Table F-24Spleen Cell Proliferative Response to Anti-CD3 Stimulation in Female Sprague Dawley Rats Exposed to Specular Hematite for Four Weeks
| Chamber Control | 15 mg/m3 | 30 mg/m3 | 60 mg/m3 | H/NH | Trend Analysis | |
|---|---|---|---|---|---|---|
| n | 8 | 8 | 8 | 8 | – | – |
| Spleen Cell Cultures | ||||||
| Unstimulated | 12,109 ± 710 | 15,842 ± 1,271* | 15,326 ± 1,226* | 13,157 ± 2,271 | NH | NS |
| Stimulated | 79,744 ± 7,899 | 119,966 ± 17,221 | 95,422 ± 10,893 | 80,043 ± 11,647 | H | NS |
| Total Spleen Cells × 107 | 59.79 ± 3.79 | 60.50 ± 4.02 | 60.71 ± 3.52 | 61.34 ± 3.44 | H | NS |
- *
Significantly different (p ≤ 0.05) from the chamber control group by Wilcoxon’s rank test.
Female Sprague Dawley rats were exposed to chamber control air or specular hematite by inhalation. On the day of euthanasia, spleens were placed into tubes containing medium and sent to Virginia Commonwealth University (Richmond, VA) on ice for next-day cell preparation. Spleens were prepared into single-cell suspensions and incubated in flat-bottom microtiter plates. The medium for the proliferative assay was RPMI supplemented with 10% FBS and 50 μM 2-mercaptoethanol. The spleen cells were cultured in either nontreated or treated anti-CD3 wells. Prior to harvest, the cells were pulsed with 3H-thymidine for 18 to 24 hours. The cultured cell data are presented as the mean ± standard error CPM derived from four replicate cultures from the number of animals indicated by n.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance; differences from the chamber control group are not significant by Dunnett’s t-test. Nonhomogeneous data were evaluated using a nonparametric analysis of variance. When significant differences occurred, exposed groups were compared to the chamber control group using Wilcoxon’s rank test. Jonckheere’s test was used to assess the significance of exposure concentration-related trends.
NS = not significant.
Table F-25Spleen Cell Proliferative Response to Anti-CD3 Stimulation in Female Sprague Dawley Rats Exposed to Specular Hematite for 26 Weeks
| Chamber Control | 15 mg/m3 | 30 mg/m3 | 60 mg/m3 | H/NH | Trend Analysis | |
|---|---|---|---|---|---|---|
| n | 8 | 8 | 8 | 8 | – | – |
| Spleen Cell Cultures | ||||||
| Unstimulated | 2,804 ± 697 | 3,902 ± 352 | 3,896 ± 304 | 3,576 ± 673 | H | NS |
| Stimulated | 85,255 ± 20,785 | 103,305 ± 8,458 | 135,331 ± 12,196 | 124,159 ± 8,030 | NH | p ≤ 0.05 |
| Total Spleen Cells × 107 | 64.61 ± 3.56 | 73.36 ± 4.02 | 64.82 ± 3.37 | 66.66 ± 2.67 | H | NS |
Female Sprague Dawley rats were exposed to chamber control air or specular hematite by inhalation. On the day of euthanasia, spleens were placed into tubes containing medium and sent to Virginia Commonwealth University (Richmond, VA) on ice for next-day cell preparation. Spleens were prepared into single-cell suspensions and incubated in flat-bottom microtiter plates. The medium for the proliferative assay was RPMI supplemented with 10% FBS and 50 μM 2-mercaptoethanol. The spleen cells were cultured in either nontreated or treated anti-CD3 wells. Prior to harvest, the cells were pulsed with 3H-thymidine for 18 to 24 hours. The cultured cell data are presented as mean ± standard error CPM derived from four replicate cultures from the number of animals indicated by n.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance. Nonhomogeneous data were evaluated using a nonparametric analysis of variance. Differences from the chamber control group are not significant by Dunnett’s t-test (homogenous data) or Wilcoxon’s rank test (nonhomogeneous data). Jonckheere’s test was used to assess the significance of exposure concentration-related trends.
NS = not significant.
Table F-26Natural Killer Cell Activity in Female Sprague Dawley Rats Exposed to Specular Hematite for Four Weeks
| Chamber Control | 15 mg/m3 | 30 mg/m3 | 60 mg/m3 | H/NH | Trend Analysis | |
|---|---|---|---|---|---|---|
| n | 8 | 8 | 8 | 8 | – | – |
| Spleen Wt. (mg) | 564 ± 26 | 526 ± 28 | 516 ± 14 | 514 ± 24 | H | p ≤ 0.05 |
| Effector:Target Ratio | ||||||
| 200:1 | 29.6 ± 1.8 | 31.6 ± 2.4 | 28.4 ± 2.7 | 32.6 ± 3.0 | H | NS |
| 100:1 | 20.4 ± 1.8 | 22.9 ± 1.4 | 20.8 ± 2.4 | 24.2 ± 2.0 | H | NS |
| 50:1 | 14.5 ± 1.2 | 17.3 ± 1.3 | 13.6 ± 1.4 | 17.5 ± 1.9 | H | NS |
| 25:1 | 9.4 ± 0.7 | 11.2 ± 0.8 | 9.8 ± 1.1 | 12.1 ± 1.5 | H | NS |
| 12.5:1 | 6.9 ± 0.9 | 8.3 ± 0.8 | 6.8 ± 0.9 | 8.5 ± 1.2 | H | NS |
| 6.25:1 | 6.7 ± 0.6 | 7.2 ± 0.4 | 6.0 ± 0.7 | 7.3 ± 0.9 | H | NS |
Female Sprague Dawley rats were exposed to chamber control air or specular hematite by inhalation. On the day of euthanasia, spleens were placed into tubes containing medium and sent to Virginia Commonwealth University (Richmond, VA) on ice for next-day cell preparation. Spleens were prepared into single-cell suspensions and were assayed 4 hours later for natural killer cell activity using 51Cr-labeled YAC-1 cells as the target. Spontaneous release over the 4-hour incubation period was 12.4% of maximum release. NK cell activity values represent the mean ± standard error percent cytotoxicity derived from the number of animals indicated by n.
H = homogeneous data and NH = nonhomogeneous data using the Bartlett test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance; differences from the chamber control group are not significant by the Dunnett t-test. The Jonckheere test was used to assess the significance of exposure concentration-related trends.
NS = not significant.
Table F-27Natural Killer Cell Activity in Female Sprague Dawley Rats Exposed to Specular Hematite for 26 Weeks
| Chamber Control | 15 mg/m3 | 30 mg/m3 | 60 mg/m3 | H/NH | Trend Analysis | |
|---|---|---|---|---|---|---|
| n | 8 | 8 | 8 | 8 | – | – |
| Spleen Wt. (mg) | 608 ± 21 | 655 ± 20 | 599 ± 35 | 644 ± 25 | H | NS |
| Effector:Target Ratio | ||||||
| 200:1 | 16.2 ± 3.9 | 23.3 ± 1.9 | 24.2 ± 1.7 | 24.6 ± 1.9 | H | p ≤ 0.05 |
| 100:1 | 9.0 ± 2.3 | 13.1 ± 1.3 | 14.7 ± 1.1 | 14.4 ± 1.7 | H | p ≤ 0.05 |
| 50:1 | 3.4 ± 1.2 | 7.1 ± 1.1 | 7.5 ± 1.0* | 7.4 ± 1.2 | H | p ≤ 0.05 |
| 25:1 | 1.3 ± 30.7 | 3.6 ± 0.7 | 4.1 ± 0.8 | 4.7 ± 1.0* | H | p ≤ 0.01 |
| 12.5:1 | <1 | 1.9 ± 0.3 | 2.2 ± 0.8 | 2.2 ± 0.7 | H | p ≤ 0.05 |
| 6.25:1 | <1 | <1 | <1 | 1.0 ± 0.6 | H | p ≤ 0.05 |
- *
Significantly different (p ≤ 0.05) from the chamber control group by Dunnett’s t-test.
Female Sprague Dawley rats were exposed to chamber control air or specular hematite by inhalation. On the day of euthanasia, spleens were placed into tubes containing medium and sent to Virginia Commonwealth University (Richmond, VA) on ice for next-day cell preparation. Spleens were prepared into single-cell suspensions and were assayed 4 hours later for natural killer cell activity using 51Cr-labeled YAC-1 cells as the target. Spontaneous release over the 4-hour incubation period was 14.3% of maximum release. NK cell activity values represent the mean ± standard error percent cytotoxicity derived from the number of animals indicated by n.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance; when significant differences occurred, exposed groups were compared to the chamber control group using Dunnett’s t-test. Jonckheere’s test was used to assess the significance of exposure concentration-related trends.
NS = not significant.
Table F-28Bronchoalveolar Lavage Fluid Cytokine Levels in Female Sprague Dawley Rats Exposed to Specular Hematite for 4 or 26 Weeks
| Chamber Control | 15 mg/m3 | 30 mg/m3 | 60 mg/m3 | H/NH | Trend Analysis | |
|---|---|---|---|---|---|---|
| n | 8 | 8 | 8 | 8 | – | – |
| Week 4 | ||||||
| IL-1α | 21.31 ± 7.12 | 27.05 ± 12.49 | 16.18 ± 4.51 | 168.20 ± 106.45 | NH | NS |
| MCP-1 | 22.12 ± 0.89 | 18.94 ± 4.43 | 22.24 ± 0.77 | 45.77 ± 13.12 | NH | p ≤ 0.05 |
| TNF-α | 12.63 ± 3.99 | 15.62 ± 6.69 | 8.97 ± 3.02 | 98.33 ± 63.37 | NH | NS |
| IFN-γ | ND | ND | ND | 27.58 ± 22.95 | NH | p ≤ 0.01 |
| GM-CSF | 45.96 ± 7.48 | 48.08 ± 15.92 | 40.75 ± 7.85 | 125.33 ± 49.24 | NH | NS |
| IL-4 | 0.04 ± 0.04 | 0.65 ± 0.34 | ND | 5.36 ± 3.89 | NH | p ≤ 0.05 |
| Week 26 | ||||||
| IL-1α | 40.49 ± 18.39 | 21.89 ± 5.42 | 6.96 ± 4.97 | 23.38 ± 15.37 | NH | p ≤ 0.01 |
| MCP-1 | 25.37 ± 2.55 | 27.06 ± 2.48 | 91.31 ± 8.88** | 782.32 ± 99.06** | NH | p ≤ 0.01 |
| TNF-α | 22.51 ± 11.00 | 10.73 ± 3.97 | 3.47 ± 2.56 | 11.60 ± 7.29 | NH | p ≤ 0.01 |
| IFN-γ | 0.25 ± 0.25 | ND | ND | ND | NH | p ≤ 0.01 |
| GM-CSF | 44.39 ± 22.24 | 43.39 ± 10.57 | 13.92 ± 9.68 | 28.72 ± 19.09 | H | p ≤ 0.01 |
| IL-4 | 1.13 ± 0.67 | ND | ND | 0.77 ± 0.56 | NH | p ≤ 0.01 |
- **
Significantly different (p ≤ 0.01) from the chamber control group by Wilcoxon’s rank test.
Female Sprague Dawley rats were exposed to chamber control air or specular hematite by inhalation. On the day of euthanasia, bronchoalveolar lavage fluid was collected and frozen. Samples were sent to Virginia Commonwealth University (Richmond, VA) on dry ice for sample analysis. Cytokine levels were analyzed in 25 μL aliquots of sample using FlowCytomixTM bead array kits according to kit instructions. Values represent the mean ± standard error pg/mL of each cytokine derived from the number of males indicated by n.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Homogeneous data were evaluated using a parametric analysis of variance; differences from the chamber control group are not significant by Dunnett’s t-test. Nonhomogeneous data were evaluated using a nonparametric analysis of variance. When significant differences occurred, exposed groups were compared to the chamber control group using Wilcoxon’s rank test. Jonckheere’s test was used to assess the significance of exposure concentration-related trends.
NS = not significant; ND = not detected.
Table F-29Bronchoalveolar Lavage Fluid Cytokine Levels in Male Sprague Dawley Rats Exposed to Specular Hematite for 4, 16, 26, or 39 Weeks
| Chamber Control | 15 mg/m3 | 30 mg/m3 | 60 mg/m3 | H/NH | Trend Analysis | |
|---|---|---|---|---|---|---|
| n | 8 | 8 | 8 | 8 | ||
| Week 4 | ||||||
| IL-1α | ND | 5.30 ± 3.82 | 16.90 ± 7.58 | 17.29 ± 13.16 | NH | p ≤ 0.05 |
| MCP-1α | 15.04 ± 0.69 | 17.06 ± 1.56 | 17.67 ± 0.91 | 18.40 ± 2.16 | NH | NS |
| TNF-α | ND | 4.65 ± 2.28 | 2.56 ± 2.20 | 4.45 ± 2.92 | NH | p ≤ 0.01 |
| IFN-γ | ND | ND | ND | ND | NA | NA |
| GM-CSF | 4.53 ± 1.98 | 32.26 ± 12.54 | 39.69 ± 10.45 | 25.61 ± 13.59 | NH | NS |
| IL-4 | ND | 0.06 ± 0.06 | ND | 0.17 ± 0.17 | NH | p ≤ 0.01 |
| Week 16 | ||||||
| IL-1α | 13.84 ± 6.58 | 18.75 ± 14.29 | 10.93 ± 10.93 | 2.15 ± 2.15 | NH | p ≤ 0.01 |
| MCP-1 | 17.60 ± 0.98 | 18.17 ± 1.89 | 33.86 ± 2.73** | 296.39 ± 40.46** | NH | p ≤ 0.01 |
| TNF-α | 3.44 ± 2.72 | ND | 2.23 ± 2.23 | ND | NH | p ≤ 0.01 |
| IFN-γ | ND | ND | ND | ND | NA | NA |
| GM-CSF | 39.93 ± 11.47 | 9.84 ± 7.86 | 18.47 ± 12.54 | 2.80 ± 2.01 | NH | p ≤ 0.01 |
| IL-4 | 0.12 ± 0.12 | ND | 0.06 ± 0.06 | ND | NH | p ≤ 0.01 |
| Week 26 | ||||||
| IL-1α | 3.70 ± 3.70 | 21.82 ± 10.97 | 8.44 ± 4.82 | 33.00 ± 9.06 | NH | p ≤ 0.01 |
| MCP-1 | 12.92 ± 0.68 | 22.49 ± 3.22** | 60.20 ± 5.78** | 394.99 ± 87.34** | NH | p ≤ 0.01 |
| TNF-α | ND | 10.14 ± 7.15 | ND | 1.04 ± 1.04 | NH | p ≤ 0.01 |
| IFN-γ | ND | ND | ND | ND | NA | NA |
| GM-CSF | 4.60 ± 4.60 | 33.04 ± 16.47 | 23.57 ± 11.16 | 16.22 ± 9.95 | NH | p ≤ 0.05 |
| IL-4 | ND | 0.09 ± 0.09 | 0.04 ± 0.04 | ND | NH | p ≤ 0.01 |
| Week 39 | ||||||
| IL-1α | 39.89 ± 14.09 | 0.36 ± 0.36 | 15.51 ± 15.51 | 9.23 ± 6.30 | NH | p ≤ 0.01 |
| MCP-1 | 19.77 ± 1.87 | 35.26 ± 10.73 | 149.95 ± 30.47** | 838.08 ± 150.36** | NH | p ≤ 0.01 |
| TNF-α | 3.88 ± 2.58 | ND | 3.58 ± 3.58 | ND | NH | p ≤ 0.01 |
| IFN-γ | ND | ND | ND | ND | NA | NA |
| GM-CSF | 53.22 ± 14.81 | 2.73 ± 2.73 | 16.35 ± 16.35 | 15.72 ± 10.47 | NH | p ≤ 0.01 |
| IL-4 | 0.51 ± 0.33 | ND | 0.43 ± 0.43 | ND | NH | p ≤ 0.01 |
- **
Significantly different (p≤ 0.01) from the chamber control group by Wilcoxon’s rank test.
Male Sprague Dawley rats were exposed to chamber control air or specular hematite by inhalation. On the day of euthanasia, bronchoalveolar lavage fluid was collected and frozen. Samples were sent to Virginia Commonwealth University (Richmond, VA) on dry ice for sample analysis. Cytokine levels were analyzed in 25 μL aliquots of sample using FlowCytomixTM bead array kits according to kit instructions.
H = homogeneous data and NH = nonhomogeneous data using Bartlett’s test for homogeneity. Nonhomogeneous data were evaluated using a nonparametric analysis of variance. When significant differences occurred, exposed groups were compared to the chamber control group using Wilcoxon’s rank test. Jonckheere’s test was used to assess the significance of exposure concentration-related trends. Values represent the mean ± standard error pg/mL of each cytokine derived from the number of animals indicated by n.
NS = not significant; ND = not detected; NA = not applicable.
Table F-30Antinuclear Antibody-Positive Responses in Sprague Dawley Rats Exposed to Specular Hematite
| Chamber Control | 15 mg/m3 | 30 mg/m3 | 60 mg/m3 | |
|---|---|---|---|---|
| n | 8 | 8 | 8 | 8 |
| Male | ||||
| Week 4 | 0 (0) | 1 (13) | 0 (0) | 0 (0) |
| Week 16 | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| Week 26 | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| Week 39 | 0 (0) | 0 (0) | 0 (0) | 1 (13) |
| Female | ||||
| Week 4 | 0 (0) | 1 (13) | 0 (0) | 0 (0) |
| Week 26 | 0 (0) | 0 (0) | 2 (25) | 0 (0) |
Male and female Sprague Dawley rats were exposed to chamber control air or specular hematite by inhalation. On the day of euthanasia, serum was collected and frozen. Samples were sent to Virginia Commonwealth University (Richmond, VA) on dry ice for sample analysis. Samples were diluted 1:50 and analyzed for the presence of antinuclear antibodies by enzyme-linked immunosorbent assay. Results are presented as the incidence (percent) of positive responses. A positive response was defined as a sample having an optical density that exceeded the upper limit of the 99% confidence interval of the mean of the chamber control group at the same time point. Differences in incidence from the chamber control group are not significant by the Fisher exact chi-square test.
Figure F-1
MCP-1 Levels in the Bronchoalveolar Lavage Fluid of Male Sprague Dawley Rats Exposed to Blasting Sand by Inhalation for 4, 16, 26, or 39 Weeks
**Significantly different (p ≤ 0.01) from the chamber control group at the same time point.
#Significantly different (p ≤ 0.05) from the week 4 time point within the same exposure concentration.
##p ≤ 0.01.
Figure F-2
MCP-1 Levels in Bronchoalveolar Lavage Fluid of Male Sprague Dawley Rats Exposed to Specular Hematite by Inhalation for 4, 16, 26, or 39 Weeks
**Significantly different (p ≤ 0.01) from the chamber control group at the same time point.
#Significantly different (p ≤ 0.05) from the week 4 time point within the same exposure concentration.
## p ≤ 0.01.
- Immunotoxicity Studies - NTP Technical Report on the Toxicity Studies of Abrasiv...Immunotoxicity Studies - NTP Technical Report on the Toxicity Studies of Abrasive Blasting Agents Administered by Inhalation to F344/NTac Rats and Sprague Dawley (Hsd:Sprague Dawley® SD®) Rats
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