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Immunotoxicity

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Description of key information

No immunotoxicity data of sufficient quality are available for tungsten trioxide (target substance). However, immunotoxicity data are available for sodium tungstate (source substance), which will be used for read across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the attached description of the read-across approach.

To determine if sodium tungstate can cause an unnatural immune response, mice were exposed in utero by parental inhalation or ingestion of an estimated dose of 1 and 4 mg/kg/d, respectively; and inoculated with respiratory syncytial virus (within 2 weeks of weaning). Compared with controls, sodium tungstate treated animals presented significant spleen enlargement (splenomegaly); however, the nature of the hematological/immunological disease was inconclusive (Fastje et al., 2012).

Osterburg et al. (2014) tested if exposure to sodium tungstate can result in immune suppression. For this, parental male and female mice were orally exposed to 2, 62.5, 125, and 200 mg/kg/d for 28 d in a one-generation model and intraperitoneally injected with Staphylococcal enterotoxin. Exposure to sodium tungstate at the highest dose resulted in a decrease of activated cytotoxic T-cells and helper T-cells. In delayed-type hypersensitivity Type IV experiments, exposure to 200 mg/kg/d of sodium tungstate prior to primary and secondary antigen challenge significantly reduced footpad swelling. According to the authors, these results suggest that sodium tungstate could cause an immune suppression that may reduce host defense against pathogens.

Kelly et al. (2013) exposed mice for 16 weeks to sodium tungstate via the drinking water. Mice receiving 15, 200, or 1000 mg/L (oral doses estimated to be in the order of 50 and 250 mg/kg-day) had a significantly greater percentage of cells in the late pro-/large pre-B developmental stages. Tungstate did not alter erythrocyte and platelet counts, or hemoglobin and hematocrit levels, and neither caused liver toxicity as assessed by peripheral blood alanine aminotransferase (AST) and aspartate aminotransferase (ALT) enzyme levels. An increase in DNA damage in both whole marrow and isolated B cells at the lower tungsten concentration (15 mg/L), but not at the highest concentration (1000 mg/L), was also noted.

A US National Toxicology Program dose range finding report (US NTP, 2012) summarizes a mice immunotoxicity 28-day study to establish the potential effects on the immune system of female mice receiving sodium tungstate via drinking water at 125, 250, 500, 1000, and 2000 mg/L (estimated doses ranged from 30 to 500 mg/kg-day). Over the 28-day exposure period, sodium tungstate did not alter body weight, body weight gain, or the weights of the major organs of the immune system, the thymus and spleen. Total splenocyte number and both absolute values and percent values of spleen cell phenotypes were unaffected by sodium tungstate exposure (NTP, 2012). No effects were observed on T-dependent antibody responses, as evaluated using the antibody-forming cell response, the sheep red blood cell enzyme-linked immunosorbent assay (ELISA), and the keyhole limpet hemocyanin ELISA, suggesting that humoral (antibody) immunity is not adversely affected by sodium tungstate exposure (US NTP, 2012). The mixed leukocyte response and the cytotoxic T-lymphocyte responses presented some significant differences, but they were not dose responsive. Furthermore, no effects were observed on thein vivodelayed-type hypersensitivity response to Candida albicans or in the anti-CD3-mediated proliferation assay, suggesting that sodium tungstate does not affect cell-mediated immunity (US NTP, 2012).

Frawley et al. (2016) published a reinterpretation of the NTP study results on female B6C3F1/N mice study. The publication indicated that three different parameters of cell-mediated immunity were similarly affected at 1000 mg/L. T-cell proliferative responses against allogeneic leukocytes and anti-CD3 were decreased. Cytotoxic T-lymphocyte activity was decreased at all effector:target cell ratios examined. At 2000 mg/L, the absolute numbers of CD3+T-cell progenitor cells in bone marrow were increased but the alterations in B-lymphocyte and other progenitor cells were not significant. There were no effects on bone marrow DNA synthesis or colony forming capabilities. Tungstate-induced effects on humoral-mediated immunity, innate immunity, and splenocyte sub-populations were limited. Enhanced histopathology did not detect treatment-related lesions in any of the immune tissues.

The following information is considered for any hazard / risk assessment:

Overall, there are drinking water immunotoxicity 28 -day studies (Osterburg et al., 2014) conducted at doses (200 -250 mg/kg/d) that reported potential effects of sodium tungstate on innate, humoral or cell mediated immunities. US NTP sponsored standard testing studies at similar exposure doses (1000 mg/L≈245 mg/kg-day) found that the tungstate induced humoral-mediated immunity, innate immunity, and splenocyte sub-populations effects were limited.

In addition, female B6C3F1/N mice exposed to sodium tungstate in the drinking water demonstrated no effects on body weight, body weight gain or the weights of major organs of the immune system, the thymus and the spleen, over the 28-day exposure period.

No effects were observed on cell viabilities [total spleen cell numbers or on the absolute or percent values of splenic B-cells, T-cells, T-cell subsets, natural killer (NK) cells, or macrophages], humoral immunity examinations [e.g. antibody forming cell assay (AFC) response to sheep blood red blood cells [SRBC] in the plaque assays or on serum IgM antibody levels to SRBC or Keyhole limpet hemocyanin(KLH)], and cell-mediated immunity (no effects on NK cell activity). Exposure to sodium tungstate did not affect DNA synthesis of bone marrow cells, or colony-forming units following in vitro stimulation of isolated bone marrow cells with macrophage colony-M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythroid colony-stimulating factor (E-CSF). Furthermore, no effects were observed on the in vivo delayed-type hypersensitivity (DTH) response to C. albicans.

The US NTP study suggests that exposure to sodium tungstate in drinking water may adversely affect cell-mediated immunity only following sensitization with an immune stimulating agent such as P815 mastocytoma cells), and as well as splenocyte proliferation against allogeneic leukocytes or the anti-CD3 antibody at 1000 mg/L only, as surprisingly, no effects were observed on cell-mediated immunity responses at 2000 mg/L (Frawley et al., 2016). Similarly, other studies have shown that tungstate-treated C57BL/6 mice exposed pup sin utero via water (15 ppm, ad libetum) and inhalation (3.33 mg/m3),and after weaning (15 ppm, ad libetum) developed significant splenomegaly when inoculated with the T-cell activating respiratory syncytial virus (RSV) (Fastje et al., 2012); and demonstrated suppression of activated TH and TCTL cells, when exposed parental (28-days) and pups (12-19 weeks after weaning) to 200 mg/kg-day and challenged with bacterial antigen staphylococcal enterotoxin (SEB) (Osterburg et al., 2014). However, in the absence of RSV or SEB challenge, tungstate had no effect on spleen size or T-cell populations.

In summary, sodium tungstate did not affect innate immunity, humoral immunity, or on developing hematopoietic cells in the bone marrow, and on unstimulated splenocyte phenotypes in female B6C3F1/N mice exposed via the drinking water. In addition, sodium tungstate did not adversely affect immune-organs of mice (thymus, spleen, mesenteric lymph node, popliteal lymph node, mucosa-associated lymphoid tissues, or bone marrow) and rats (McInturf et al, 2011). As potential cell-mediated immune effects at 1000 mg/L (≈dose level 247 mg/kg/d) are only observed under conditions of co-exposure to an immune-stimulating agent (such as tumor cells or genetically dissimilar leukocytes) rather than direct action. Based on this, sodium tungstate is not considered a direct immunotoxicant.

Key value for chemical safety assessment

Effect on immunotoxicity: via oral route

Link to relevant study records

Referenceopen allclose all

Endpoint:
immunotoxicity: sub-chronic oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium tungstate
Target: Tungsten Trioxide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Reason / purpose:
read-across: supporting information
Reason / purpose:
reference to same study
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Sodium tungstate dihydrate (STD, Na2WO4 2H2O, CAS #10213-10-2, Lot #12330JO)
Species:
mouse
Strain:
B6C3F1
Sex:
female
Details on test animals and environmental conditions:
Female pathogen-free B6C3F1/N mice were obtained at 4–8 weeks of age and maintained on a 12-h light/dark cycle at 18–26 C
Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
The mice were exposed to STD via the drinking water at 125, 250, 500, 1000, or 2000 mg/L for 28 d and through the morning of Day 29 until the time of study termination.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The 125, 1000, and 2000 mg STD/L dose formulations were analyzed using ion chromatography. The concentrations of all dose formulations tested were within 10% of the target, the NTP acceptance limit
Duration of treatment / exposure:
28 days
Frequency of treatment:
Exposed daily for 28-days via dringking water
Dose / conc.:
125 mg/L drinking water
Remarks:
Approximately 30 mg/kg/day
Dose / conc.:
250 mg/L drinking water
Remarks:
Approximately 60 mg/kg/day
Dose / conc.:
500 mg/L drinking water
Remarks:
Approximately 125 mg/kg/day
Dose / conc.:
1 000 mg/L drinking water
Remarks:
Approximately 250 mg/kg/day
Dose / conc.:
2 000 mg/L drinking water
Remarks:
Approximately 500 mg/kg/day
No. of animals per sex per dose:
8 animals per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- The doses, vehicle, and route of exposure were selected for consistency with the 13- week NTP mouse toxicology studies, and were based on mechanistic immunotoxicology studies conducted by the Naval Health Research Center Detachment Environmental Health Effects Laboratory (NHRC Det) (Osterburg et al. 2014)

- The average (N ¼ 5) determined concentration for the 125 mg STD/L formulation was 123.4 mg STD/L, the average for the 1000 mg STD/L formulation was 977.8 mg STD/L, and the average for the 2000 mg STD/L formulation was 1870 mg
STD/L. At 1000 and 2000 mg/L, STD formed a white precipitate following the initial dissolution in tap water.
Observations and clinical examinations performed and frequency:
- Mice were weighed prior to initiation of the study, and on Days 1, 8, 15, 22 and 29
Sacrifice and pathology:
- Organ weights were obtained for the liver, spleen, lungs, thymus, kidneys and adrenal glands
- Hematology parameters evaluated included: reticulocytes, erythrocyte and leukocyte numbers, leukocyte differentials, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelet number.
- At necropsy, liver, spleen, lungs, thymus, kidneys, adrenals, bone marrow (femur), gastrointestinal (GI) tract were collected.
Humoral immunity examinations:
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA): Yes
- Method: Anti-keyhole limpet hemocyanin (KLH) antibody ELISA
Specific cell-mediated immunity:
DELAYED-TYPE HYPERSENSITIVITY (DTH) REACTION: Yes / No / No data
- The DTH response to C. albicans was conducted

CYTOTOXIC T-LYMPHOCYTE (CTL) ASSAY: Yes
- The cytotoxic activity of TCTL cells (CTL activity) against P815 mastocytoma cells was conducted
Non-specific cell-mediated immunity:
NATURAL KILLER (NK) CELL ACTIVITY: Yes
- NK-cell activity was assessed using [Cr]-labeled YAC-1 cells as the target for NK-mediated cytotoxicity
Other functional activity assays:
SPLEEN CELL PROLIFERATION ASSAY (ANTI-CD3 MEDIATED T CELL PROLIFERATION)
T-cell proliferation following stimulation with anti-CD3 antibody was evaluated using Biocoat T-cell activation plates. Samples were counted and [3H]-thymidine incorporation into proliferating cells was used as the assay endpoint.

ENUMERATION TOTAL B CELLS, TOTAL T CELLS AND T CELL SUBPOPULATIONS
Single-cell suspensions of splenocytes were analyzed by flow cytometry to quantify various cell populations. The following cell populations were evaluated: B-lymphocytes (B-cells, Igþ ), total T-lymphocytes (T-cells, CD3þ ), T-cell subsets (CD4þ CD8. , CD4. CD8þ and CD4þ CD8þ ), NK cells (NK1.1þ CD3. ) and macrophages.

OTHER ASSAYS [SPECIFY IF APPLICABLE]: Functional activity of the mononuclear phagocytic system (MPS)
Positive control:
- Positive controls used in these studies included rabbit antiasialo GM1 antibody (AAGM1) for natural killer (NK) cell activity, maleic vinyl ether (MVE) was the positive control for mononuclear phagocytic system (MPS) activity, and cyclophosphamide (CPS) was the positive was the positive control for all other assays
Statistics:
Homogeneous data were evaluated using analysis of variance (ANOVA) and Dunnett’s test. Non-homogeneous data were evaluated using Welch ANOVA and, for pairwise comparisons with the control group, Wilcoxon Rank Sum Tests or Dunn’s test (bone marrow data). Student’s t-test was used to compare the
vehicle and positive control groups, and for the DTH challenge only group. Jonckheere’s test was used to test for dose related trends. Data that were different from control at two-sided p<0.05 and trends with one-sided p 0.05 were considered statistically significant.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no significant differences in body weights throughout the study, or in overall body weight gain (Day 29–Day 1), between treated and control animals.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
With the exception of a 9% decrease (p<0.01) for animals in the 2000 mg/L exposure group at the 1-week timepoint, there were no significant differences in water consumption between control- and sodium tungstate exposed mice
Ophthalmological findings:
not specified
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- No effects were observed on the absolute or relative weights of these organs in any of the studies, with two exceptions. In the hematology study, an increase
(11%, p 0.01) in relative liver weight at 2000 mg STD/L was observed. In the MPS study, an increase (9%, p 0.05) in spleen weight at 250 mg STD/L was noted. These effects were not observed in the remaining studies and thus, were not considered to be biologically significant.
- The absolute numbers of specific blood leukocyte populations were unaffected by STD exposure. However, when the leukocyte differentials were evaluated on a percent basis, neutrophils were decreased 17–33% in all tungstate treated groups, and lymphocyte. were increased 14% at 250–1000 mg/L, relative to the vehicle control. In addition, monocytes were decreased (15% and 21%) with 500 and 1000 mg/L. No effects were observed on blood hematology parameters, with the exception of MCH and MCHC. MCH was increased (4%) at 125 mg/L, and decreased (4%) at 250 and 500 mg STD/L; MCHC was decreased 5% with 500 mg/L.
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
effects observed, treatment-related
Description (incidence and severity):
xposure to STD in drinking water for 28 d at doses
of 125–2000 mg/L had limited effect on humoral and innate
immunity, on developing hematopoietic cells in the bone marrow,
and on unstimulated splenocyte phenotypes in B6C3F1/N mice.
However, such exposure may have decreased the functional activity
of T-lymphocytes at 1000 mg STD/L, as evidenced by the
reduction of TCTL activity, and the proliferative response to allogeneic
leukocytes and the anti-CD3 antibody, while increasing the
activity at lower doses. These data indicated that, under conditions
of co-exposure to an immune-stimulating agent, such as
tumor cells or genetically dissimilar leukocytes, STD may modulate
the normal cell-mediated immune response.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Weights of the liver, spleen, thymus, lungs, and kidneys were measured as a component of three studies, the MPS study (positive control), the hematology study, and the collection of tissues for histopathology. No effects were observed on the absolute or relative weights of these organs in any of the studies, with two exceptions. In the hematology study, an increase (11%, p<0.01) in relative liver weight at 2000 mg/L was observed. In the MPS study, an increase (9%, p 0.05) in spleen weight at 250 mg/L was noted. These effects were not observed in the remaining studies and thus, were not considered to be biologically significant.

Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related lesions were detected in the thymus, spleen, mesenteric lymph node, popliteal lymph node, mucosa-associated lymphoid tissues, or bone marrow
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related lesions were detected in the thymus, spleen, mesenteric lymph node, popliteal lymph node, mucosa-associated lymphoid tissues, or bone marrow
Histopathological findings: neoplastic:
not specified
Cell viabilities:
no effects observed
Description (incidence and severity):
No effects on the total spleen cell numbers or on the absolute or percent values of splenic B-cells, T-cells, T-cell subsets, NK cells, or macrophages were observed following 28 d of exposure to sodium tungstate
Humoral immunity examinations:
no effects observed
Description (incidence and severity):
No sodium tungstate exposure-related effects were observed on the AFC response to SRBC in the plaque assays or on serum IgM antibody levels to SRBC or KLH
Specific cell-mediated immunity:
effects observed, treatment-related
Description (incidence and severity):
- Teatment with 1000 mg/L resulted in decreased ex vivo activity of splenic T-CTL cells against P815 mastocytoma cells, relative to activity resulting from host exposure to the vehicle control, at all effector:target (E:T) ratios examined.
- At 500 mg/L, the cytotoxic activity of T-CTL cells was increased. No effects were observed with 125, 250, or 2000 mg/L.
- A decrease (32%) in ex vivo leukocyte proliferation in response to stimulation by allogeneic leukocytes was noted following host exposure to 1000 mg/L, with an increase in activity (32%) at 250 mg/L. The response of the cells from animals in the high-dose group (2000 mg/L) was not different from that observed in the vehicle control mice cells. When stimulated with anti-CD3 antibody, splenocyte cell proliferation was decreased 21% with 1000 mg /L, although this point was not statistically significant.
- The basal proliferative response in those cultures was decreased 11% at 125 mg/L, and increased 32% at 500 mg/L. No effects were observed on the delayed-type hypersensitivity response to C. albicans
Non-specific cell-mediated immunity:
no effects observed
Description (incidence and severity):
- No effects on NK cells, were observed following 28 d of exposure to sodium tungstate.
- No significant effect on NK cell activity was noted in any sodium tungstate treatment group.
Description (incidence and severity):
Innate Immunity:
- There was no alteration in the ability of fixed-tissue macrophages to phagocytize SRBC in liver, spleen, lung, or thymus.
- Significant decreases were observed in the uptake of [51Cr]-SRBC, with the 125 (percent) and 2000 (percent and specific activity) mg/L regimens.
- Natural killer cell activity was measured at E:T ratios of 6.25:1–200:1 for all doses (N ¼ 8); no significant effect on NK cell activity was noted in any treatment group.
Other findings:
effects observed, treatment-related
Description (incidence and severity):
Exposure to sodium tungstate in the drinking water induced a 41% increase in total bone marrow cell numbers in the immunophenotyping study, but not in the CFU study. In the immunophenotyping study, the absolute values of CD3þ (T-cell lineage, 86%), B220+ (B-cell lineage, 53%), CD11b+ (granulocyte/macrophage lineage, 36%), TER-119+ (erythroid lineage, 50%), and intermediate (22%), high (35%), and total (31%) Gr1+ (neutrophils, myelocytes, and immature progenitors) cells were also increased, relative to the control, after host exposure to 2000 mg/L. However, only the CD3þ data were statistically significant. There was no effect on DNA synthesis of bone marrow cells, or colony-forming units following in vitro stimulation of isolated bone marrow cells with CSF-M, CSF-GM, or CSF-E
Dose descriptor:
LOAEL
Effect level:
1 000 mg/L drinking water
Based on:
test mat.
Sex:
female
Basis for effect level:
immunology
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
immunology
Conclusions:
The objective of this study was to assess potential immunotoxicity of sodium tungstate dihydrate (STD), a drinking water contaminant. Female B6C3F1/N mice received 0–2000 mg STD/L in their drinking water for 28 d, and were evaluated for effects on immune cell populations in spleen and bone marrow, and humoral-mediated, cell-mediated, and innate immunity. Three different parameters of cell-mediated immunity were similarly affected at 1000 mg STD/L. T-cell proliferative responses against allogeneic leukocytes and anti-CD3 were decreased 32%, and 21%, respectively. Cytotoxic T-lymphocyte activity was decreased at all effector:target cell ratios examined. At 2000 mg STD/L, the absolute numbers of CD3+ T-cell progenitor cells in bone marrow were increased 86%, but the alterations in B-lymphocyte and other progenitor cells were not significant. There were no effects on bone marrow DNA synthesis or colony forming capabilities. STD-induced effects on humoral-mediated immunity, innate immunity, and splenocyte sub-populations were limited. Enhanced histopathology did not detect treatment-related lesions in any of the immune tissues. These data suggest exposure to STD in drinking water may adversely affect cell-mediated immunity.

In summary, sodium tungstate did not affect innate immunity, humoral immunity, or on developing hematopoietic cells in the bone marrow, and on unstimulated splenocyte phenotypes in female B6C3F1/N mice exposed via the drinking water. In addition, sodium tungstate did not adversely affect immune-organs of mice (thymus, spleen, mesenteric lymph node, popliteal lymph node, mucosa-associated lymphoid tissues, or bone marrow) and rats (McInturf et al, 2011). As potential cell-mediated immune effects at 1000 mg/L (≈dose level 247 mg/kg-day) are only observed under conditions of co-exposure to an immune-stimulating agent rather than direct action.
Executive summary:

No immunotoxicity data of sufficient quality are available for tungsten trioxide (target substance). However, immunotoxicity data are available for sodium tungstate (source substance), which will be used for reading across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

Endpoint:
immunotoxicity: short-term oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Scientfically sound study similar to OECD guidelines. However as this study is used in the context of a read across, Klimisch 2 is assigned.
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium tungstate
Target: Tungstic Trioxide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Reason / purpose:
read-across: supporting information
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.7800
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
female
Route of administration:
oral: drinking water
Vehicle:
water
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Five STD concentrations (125, 250, 500, 1000 and 2000 mg/L) were utilized and administered for 28 days via the drinking water. STD solutions in tap water were freshly prepared every two weeks, and stock solutions were stored refrigerated
Duration of treatment / exposure:
Sodium tungstate administered for 28 days via the drinking water
Frequency of treatment:
Daily
Dose / conc.:
125 mg/L drinking water
Remarks:
Doses / Concentrations:
125 mg/L
Basis:
nominal in water
Dose / conc.:
250 mg/L drinking water
Remarks:
Doses / Concentrations:
250 mg/L
Basis:
nominal in water
Dose / conc.:
500 mg/L drinking water
Remarks:
Doses / Concentrations:
500 mg/L
Basis:
nominal in water
Dose / conc.:
1 000 mg/L drinking water
Remarks:
Doses / Concentrations:
1000 mg/L
Basis:
nominal in water
Dose / conc.:
2 000 mg/L drinking water
Remarks:
Doses / Concentrations:
2000 mg/L
Basis:
nominal in water
No. of animals per sex per dose:
No data
Control animals:
yes, concurrent vehicle
Humoral immunity examinations:
ANTIBODY PLAQUE FORMING CELLS (PFC) ASSAY: Yes
- Method: T-Dependent antibody response (TDAR) function :anti-sheep red blood cell plaque-forming cell (PFC) assay,

ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA): Yes
- Method: Anti-keyhole limpet hemocyanin (KLH) antibody ELISA
Specific cell-mediated immunity:
DELAYED-TYPE HYPERSENSITIVITY (DTH) REACTION: Yes

CYTOTOXIC T-LYMPHOCYTE (CTL) ASSAY: Yes
Non-specific cell-mediated immunity:
NATURAL KILLER (NK) CELL ACTIVITY: Yes
- Method: NK-cell activity was assessed using [Cr]-labeled YAC-1 cells as the target for NK-mediated cytotoxicity

OTHER ASSAYS: Tes
- Method: Functional activity of the mononuclear phagocytic system (MPS).
Other functional activity assays:
OTHER ASSAYS [SPECIFY IF APPLICABLE]: Yes
- Method: Method: Functional activity of the mononuclear phagocytic system (MPS).

Clinical signs:
not specified
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Female B6C3F1/N mice exposed to sodium tungstatein the drinking water demonstrated no effects on body weight, or body weight gain over the 28-day exposure period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
no effects observed
Description (incidence and severity):
Female B6C3F1/N mice exposed to sodium tungstate (STD) in the drinking water demonstrated no effects on body weight, body weight gain or the weights of major organs of the immune system, the thymus and the spleen, over the 28-day exposure period. Total splenocyte number and both absolute values and percent values of spleen cell phenotypes were unaffected by STD exposure. No effects were observed on T-dependent antibody responses (TDAR), as evaluated using the antibody-forming cell (AFC) response, the sheep red blood cell (sRBC) enzyme-linked immunosorbent assay (ELISA), and the keyhole limpet hemocyanin (KLH) ELISA, suggesting that STD exposure does not adversely affect humoral immunity. Although some significant differences were observed in two ex vivo cell-mediated assays (i.e. the mixed leukocyte response [MLR] and the cytotoxic T-lymphocyte [CTL] response), these differences were not dose-responsive. Furthermore, no effects were observed on the in vivo delayed-type hypersensitivity (DTH) response to C. albicans or in the anti-CD3 mediated proliferation assay, which are two additional assays used to evaluate cell-mediated immunity, suggesting overall that drinking water exposure to STD does not affect cell-mediated immunity. Finally, innate immunity was not affected by STD exposure, as indicated by a lack of effect on both natural killer (NK) cell activity and the functional activity of the mononuclear phagocytic system (MPS).
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Female B6C3F1/N mice exposed to STD in the drinking water demonstrated no effects on the weights of major organs of the immune system, the thymus and the spleen, over the 28-day exposure period. Total splenocyte number and both absolute values and percent values of spleen cell phenotypes were unaffected by STD exposure.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Female B6C3F1/N mice exposed to sodium tungstatein the drinking water demonstrated no effects on the weights of major organs of the immune system, the thymus and the spleen, over the 28-day exposure period.
Neuropathological findings:
not specified
Histopathological findings: neoplastic:
not specified
Humoral immunity examinations:
no effects observed
Description (incidence and severity):
No effects were observed on T-dependent antibody responses (TDAR), as evaluated using the antibody-forming cell (AFC) response, the sheep red blood cell (sRBC) enzyme-linked immunosorbent assay (ELISA), and the keyhole limpet hemocyanin (KLH) ELISA.
Specific cell-mediated immunity:
no effects observed
Description (incidence and severity):
No effects were observed on the in vivo delayed-type hypersensitivity (DTH) response to C. albicans or in the anti-CD3 mediated proliferation assay, which are two additional assays used to evaluate cell-mediated immunity.
Non-specific cell-mediated immunity:
no effects observed
Description (incidence and severity):
Innate immunity was not affected by STD exposure, as indicated by a lack of effect on both natural killer (NK) cell activity and the functional activity of the mononuclear phagocytic system (MPS).
Other findings:
effects observed, treatment-related
Description (incidence and severity):
When evaluated as percent values, bone marrow cell differentials were unaffected overall, with the exception of an increase in TER-119+cells (i.e. erythroid lineage cells) at the 2000 mg/L sodium tungstate exposure level
Dose descriptor:
NOAEL
Effect level:
2 000 mg/L drinking water
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'
Conclusions:
In summary, with the exception of effects on bone marrow differentials at the 2000 mg/L dose level, sodium tungstate did not adversely affect, innate immunity, humoral immunity or cell-mediated immunity in female B6C3F1/N mice exposed via the drinking water
Executive summary:

No immunotoxicity data of sufficient quality are available for tungsten trioxide (target substance). However, immunotoxicity data are available for sodium tungstate (source substance), which will be used for reading across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

Endpoint:
immunotoxicity: chronic oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Well documented scientfically study with sufficient information provided on materials and methods to evaluate results. However as this study is used in the context of a read across, Klimisch 2 is assigned.
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium tungstate
Target: Tungsten Trioxide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Reason / purpose:
read-across: supporting information
Qualifier:
no guideline followed
Principles of method if other than guideline:
Experiments were conducted as described in Current Protocols in Immunology
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
C57BL
Sex:
male/female
Details on test animals and environmental conditions:
C57BL6 (28-day study male 8–12-week-old, 19–22 g; one-gen study female 8–12-week-old, 15–18 g). Mice were allowed to acclimatize to the animal facility for 7 days before commencement of the experimental phase. During the course of the experiment, animals had a 12 h day/night cycle in a temperature-controlled room (22 C). All animals had ad libitum access to filtered water and a low molybdenum diet.
Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
Water consumption was determined using graduated water bottles. Measurements were made daily of water consumed by mice. The tungstate in the water bottles was administered to calculate approximate ingested doses of tungstate based on an estimated water consumption of 4.5 ml/mouse/day. Water bottles were changed 2–3 times weekly, always using water from the source and a 1 M sodium tungstate stock supply.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
For the 28-day study, mice were administered 0, 62.5, 125, or 200 mg/kg/day of tungstate for the course. In the one-gen study parental mice were dosed with 0, 2, 62.5, 125, or 200 mg/kg/day of tungstate. Both P and F1 mice were maintained on these doses for the course of the study.

In the one-gen exposure, mice were exposed to tungstate for 90 days prior to mating (Weeks 1–12). The next 7 weeks comprised gestation and weaning (Weeks 13–19). After pups (F1) were weaned, the parents (P) were necropsied as described, &19 weeks after initiation of the study. The F1 generation was exposed to tungstate for a further 90 days after weaning and then necropsied.
Frequency of treatment:
Daily
Dose / conc.:
0.2 mg/kg bw/day (nominal)
Remarks:
Basis:
nominal in water
Dose / conc.:
2 mg/kg bw/day (nominal)
Remarks:
Basis:
nominal in water
Dose / conc.:
62.5 mg/kg bw/day (nominal)
Remarks:
Basis:
nominal in water
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
Basis:
nominal in water
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Basis:
nominal in water
Control animals:
yes, concurrent vehicle
Sacrifice and pathology:
Animals were euthanized by CO2 asphyxiation and blood was obtained by cardiac puncture with a 23-gauge needle and placed into Na2EDTA-anti-coagulantcoated tubes. Tissues were harvested according to normal procedures, and the spleens and blood were processed for subsequent flow cytometric staining and analyses.
Humoral immunity examinations:
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA): Yes, standard ELISA for IL-6, TNFa, IL-10, and IFNg
Specific cell-mediated immunity:
DELAYED-TYPE HYPERSENSITIVITY (DTH) REACTION:

4-hydroxy-3-nitrophenylacetic acid active ester (NP-O-Su) was solubilized in 2% dimethyl sulfoxide for administration of 50 ml as a dorsal subcutaneous injection. This was immediately followed by 0.1 ml of borate-buffered saline solution (pH 8.6) to enhance haptenization and primary immune responses. During the sensitization phase, appropriate tungstate doses continued to be administered to the treated mice. Ten days later, the mice were anesthetized with 3.5% isofluorane for injection of 20 ml of NP-O-Su mixed (1:20) with PBS (pH 7.8) into the right hind foot pad with a 28-gauge needle; an equivalent volume of PBS was injected into the contralateral foot. The extent of footpad swelling was measured with a dial gauge 24 h post-injection.

Twenty-four hours prior to necropsy, mice were injected intraperitoneally (IP) with either sterile saline or 20 mg of Staphylococcal enterotoxin B in saline to induce an immune response.
Statistics:
All statistical tests were performed with Systat . Statistical significance was assumed at p <0.05. Comparisons between treatments (tungstate dose, immune challenge) were performed as two-way analysis ofnvariance (ANOVA). Differences in weights were determined by repeated measures analysis of variance.
Clinical signs:
not specified
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No statistically significant changes in body weight due to any tungstate dose levels in the 28-day, one-gen, or delayed-Type IV hypersensitivity experiments. The 200 mg/kg/day males in the P generation show a consistent trend towards decreased weights. This observation, however, was not statistically significant
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No significant changes in water consumption due to the quantity of tungstate present in the water
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Hematological parameters from the blood of animals at necropsy. With two exceptions (monocyte% and red blood cell distribution width), there were no statistical differences in the data between P and F1; therefore, the data for P and F1.
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
effects observed, treatment-related
Description (incidence and severity):
- No significant changes in any of the parameters measured in response to tungstate, with the exception of the percent monocytes (%MO). There were fewer lymphocytes in the F1 generation compared to the P generation (p<0.023), but this was not dose-related. Additionally, the red blood cell distribution width (RDW) was higher in the P generation vs the F1 pups (p50.004). The percentage of monocytes was dose-dependently lower at higher concentrations when compared to control (p<0.003). Other parameters suggest a dose-dependent trend (e.g. hematocrit); however, these trends were not statistically significant.
- Tungstate-dependent changes in either the 28-day or one-gen exposures were only observed in the spleens of animals. Furthermore, any statistically significant differences between the innate or immune responses of P and F1 mice were not noted.
- Tungstate exposure for 28 days did not result in significant changes in the percentage of helper Tcells (TH; CD3þ CD4þ ). However, the number of activated helper T-cells (CD3+, CD4+, CD71+ ), or CD71+ (transferrin receptor) TH cells were significantly reduced at the 200 mg/kg/day dose as compared to in the controls.
- Consistent with these data, the one-gen exposures resulted in reduced quantities of CD71+ TH cells in the parental mice exposed to 200 mg/kg/day. No statistically significant differences were noted in the overall quantity of CD3+, CD4+ TH cells. Additionally, there were no significant effects of generation on the suppression of CD71+ TH cells in SEB-treated mice exposed to tungstate.
- At highest tungstate group a significant reduction occurred in the quantity of activated cytotoxic T-cells (CD3+, CD8+, CD71+) in the spleens of tungstate-treated mice. The number of CD71+ CD8 cytotoxic T-cells was significantly reduced in the SEB-treated mice at high tungstate doses.
- In delayed-type hypersensitivity Type IV experiments, tungstate exposure prior to primary and secondary antigen challenge significantly reduced footpad swelling at 20 and 200 mg/kg/day.
- A dose-dependent quantitative decrease in interferon (IFN)-g levels in SEB-treated mice. In the 28-day study there was a decrease from the control mice to near baseline levels in the high-tungstate dose hosts. In a similar manner, the P mice of the one-gen exposure had a reduction from. Although not statistically significant, the F1 mice had an overall reduced IFNg response, especially at the 62.5 mg/kg/day dose, compared to their parents.
Humoral immunity examinations:
effects observed, treatment-related
Description (incidence and severity):
In the 28-day study there was a decrease from the control mice to near baseline levels in the high-tungstate dose hosts
Specific cell-mediated immunity:
effects observed, treatment-related
Description (incidence and severity):
Tungstate exposure prior to primary and secondary antigen challenge significantly reduced footpad swelling at 20 and 200 mg/kg/day.
Other functional activity assays:
effects observed, treatment-related
Description (incidence and severity):
Number of activated helper T-cells (CD3+ CD4+ CD71+ ), or CD71+ (transferrin receptor) TH cells were significantly reduced at the 200 mg/kg/day dose as compared to in the control.
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Effect level:
2 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Taken together, the data from the current studies clearly indicate tungstate exposure could result in suppression of adaptive immunity. Our data indicates little to no effect of tungstate at any dose on groups of mice exposed only to tungstate without being co-exposed to an immune stressor (e.g. SEB or DTH). This may suggest that some biological processes, such as T-cell activation, are more sensitive to tungstate.

Conclusions:
The data here suggested that tungstate exposure by oral routes could result in immunologic outcomes wherein host defense was reduced. The dose required for immune suppression in the SEB models (200 mg/kg/day) was very high; it is unlikely that this dose will be encountered through environmental sources. In contrast, in the DTH model, the dose required to observe an effect was lower with 200 and 20 mg/kg/day of tungstate reducing host immune responses. These values were approximately equivalent to exposures of 1000 and 100 ppm W, respectively. Exposure to these levels is unlikely due to environmental sources, which are substantially lower (51 ppm W).
Executive summary:

No immunotoxicity data of sufficient quality are available for tungsten trioxide (target substance). However, immunotoxicity data are available for sodium tungstate (source substance), which will be used for reading across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
132.11 mg/kg bw/day
Study duration:
subchronic
Species:
mouse
Quality of whole database:
Scientfically sound study similar to OECD guidelines. However as this study is used in the context of a read across, Klimisch 2 is assigned.

Effect on immunotoxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
immunotoxicity: short-term inhalation
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Well documented scientfically study with sufficient information provided on materials and methods to evaluate results. However as this study is used in the context of a read across, Klimisch 2 is assigned.
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium tungstate
Target: Tungsten Trioxide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Reason / purpose:
read-across: supporting information
Qualifier:
no guideline followed
Principles of method if other than guideline:
The objective of the study was to expose C57BL/6J mice to tungsten and administering a viral challenge. The Respiratory Syncytial Virus (RSV) was selected for this challenge because the protein product of the G gene possesses the FXXKXFXXA/V peptide motif within two T-cell epitopes which are restricted by
the HLA-DP2/DP4 supertype significantly associated with Pre-B ALL diagnosed between the ages of 3 and 6 years old.
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
C57BL
Sex:
male/female
Details on test animals and environmental conditions:
Eight week-old, C57BL/6J male and female mice were purchased through an IACUC approved protocol. After acclimating to the new environment, a transponder was injected at the nape of the neck of each female mouse and each mouse randomly assigned to an exposure group. The mice were housed, cared for and bred in sterile, microisolation conditions.
Route of administration:
inhalation: aerosol
Vehicle:
water
Mass median aerodynamic diameter (MMAD):
5 µm
Remarks on MMAD:
A 1% deposition rate for the target particles less than 5um (PM5) was utilized in calculating the concentration of the exposure solution
Details on exposure:
The dams were exposed to sodium tungstate aerosol 45-min, 5 days/week aerosol exposures. Female mice were exposed to a 187 g/L solution, using vacuum-drawn through a 24-port, nose-only INTOX inhalation chamber (Albuquerque, NM, USA) with an attached cascade impactor for 1 week prior to conception and 3 weeks of gestation until parturition halted exposures. Pups were weaned onto Na2WO4-spiked water (15 ppm, ad libetum).
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Female mice were exposed for 1 week prior to conception and 3 weeks of gestation until parturition halted exposures.
Frequency of treatment:
45-min, 5 days/week aerosol exposures
Water ad libitum
Dose / conc.:
33 mg/m³ air (nominal)
Remarks:
The dams were exposed to sodium tungstate (Na2WO4.2H2O) through water (15 ppm, ad libetum) and aerosol.
Dose / conc.:
15 ppm (nominal)
Remarks:
- In utero via water (15 ppm, ad libetum)
- Pups were weaned onto Na2WO4-spiked water (15 ppm, ad libetum).
No. of animals per sex per dose:
A total of 8–10 mouse pups were obtained for each group
Control animals:
yes, concurrent vehicle
Details on study design:
- At 21–35 days of age the mouse pups were lightly anesthetized and the nasal cavity inoculated with 10 uL of human RSV in medium for a total exposure of 1 106 pfu.
- Peripheral hematology was evaluated utilizing complete blood counts.

Observations and clinical examinations performed and frequency:
- Spleen tissue was massed and splenic ratio calculated as spleen mass per body mass. Spleens and femurs from each group were preserved in 10% formalin and submitted to Tissue Acquisition and Cellular/Molecular Analysis.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Statistical analysis was performed with a one-tailed Mann–Whitney test utilizing Minitab. Data are presented as medians with interquartile ranges and outliers indicated. Significance was accepted when p <0.05.
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Sodium tungstate + RSV Mouse (female) expired prior to blood draw
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- Na2WO4-only, RSV-only and Na2WO4+ RSV demonstrated significantly elevated neutrophil counts as compared to the longitudinal control. However, the first and third quartiles for all experimental groups were within the normal range except for RSV-only.
- The percentage of neutrophils contributing to the total WBC count was significantly greater for all three exposure groups as compared to the controls. However, the first and third quartiles for all experimental groups were within the normal range except for RSV-only.
- The percentage of neutrophils contributing to the total WBC count was significantly greater for all three exposure groups as compared to the controls. However, the first and third quartiles for all experimental groups were within the normal range except for RSV-only. While the percentage of neutrophils increased significantly, there was no associated increase in WBC counts for mice exposed to RSV-only.
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
effects observed, treatment-related
Description (incidence and severity):
- Na2WO4-only, RSV-only and Na2WO4+ RSV demonstrated significantly elevated neutrophil counts as compared to the longitudinal controls. However, the first and third quartiles for all experimental groups were within the normal range except for RSV-only
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
the splenomegaly present in a subset of mice in the Na2WO4+ RSV group. Splenic ratios for mice exposed to Na2WO4-only or RSV-only did not vary significantly from the controls or from each other. Splenic ratios for Na2WO4 + RSV mice were significantly larger as compared to all other groups.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Longitudinal controls and tungsten mice did not exhibit pathological indicators. When the RSV inoculation was combined with exposure to tungstate significant splenomegaly resulted.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Spleen and bone marrow tissues from two Na2- WO4+ RSV mice exhibiting the greatest degree of splenomegaly and compared with a longitudinal control mouse.
- The control exhibited a 1:1 ratio of erythropoietic and granulopoietic precursor cells with all stages represented. Both Na2WO4+ RSV mice demonstrated consistent histology, a 1:20 ratio of erythropoietic to granulocytic precursors with all stages represented. Additionally, marked thrombopoiesis was reported in both spleen and bone marrow tissues.
- No pathology was observed in the Na2WO4-only mice in this study.

Histopathological findings: neoplastic:
not specified
Details on results:
- The splenomegaly present in a subset of mice in the Na2WO4+ RSV group was accompanied by true neutrophilia, progressive anemia, and morbidity/death. Splenic ratios for mice exposed to Na2WO4-only or RSV-only did not vary significantly from the controls or from each other. Splenic ratios for Na2WO4 + RSV mice were significantly larger as compared to all other groups
- The percentage of neutrophils contributing to the total WB count was significantly greater for all three exposure groups as compared to the controls.

- While the percentage of neutrophils increased significantly, there was no associated increase in WBC counts for mice exposed to RSV only.
- No group demonstrated a significant increase in WBC counts as compared to the controls or to each other.
Cell viabilities:
not specified
Humoral immunity examinations:
not specified
Specific cell-mediated immunity:
not specified
Non-specific cell-mediated immunity:
not specified
Other functional activity assays:
not specified
Key result
Dose descriptor:
conc. level:
Effect level:
15 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Post-natal exposure to RSV can promote shift neutrophilia, but not significantly different from Na2WO4-only or Na2WO4+ RSV with most hematological measures remaining within normal parameters
Remarks on result:
not determinable

The primary, statistically significant result in this study is the occurrence of splenomegally as a result of combined exposure to both Na2WO4 and RSV. Pathological presentation associated with the splenomegaly varied, primarily presenting as true neutrophilia, but also as lymphocytosis and monocytosis. The significantly elevated neutrophil counts as compared to the controls were within the normal range. No pathology was observed in the Na2WO4-only mice in this study.

Conclusions:
This study reports the results of exposing C57BL/6J mice to Na2WO4 in utero via water (15 ppm, ad libetum) and inhalation (mean concentration PM5 3.33 mg/m3 ) and to Respiratory Syncytial Virus (RSV) within 2 weeks of weaning. Inoculation of C57BL/6J mice with RSV was associated with a neutrophil shift in 56% of 5-month old mice. When the RSV inoculation was combined with Na2WO4-exposure, significant splenomegaly resulted in addition to other hematological pathologies which were not significant.
Executive summary:

No immunotoxicity data of sufficient quality are available for tungsten trioxide (target substance). However, immunotoxicity data are available for sodium tungstate (source substance), which will be used for reading across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

Endpoint conclusion
Endpoint conclusion:
no study available

Effect on immunotoxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No (developmental) immunotoxicity data of sufficient quality are available for tungsten trioxide (target substance).Bioaccessibility studies(IITRI 2010), indicate that the bioavailability of tungsten trioxide via the oral route is limited when compared to sodium tungstate. Therefore, the read-across from sodium tungstate (source substance) to tungsten trioxide (target substance) is an appropriate conservative read-across approach for systemic toxicity. For details on the tungstate read across approach category.

No need for a Cohort 3 is needed based on two (developmental) immunotoxicity studies (Fastje et al. 2012;Osterburg et al. 2014)that exposed mice to tungstateinutero. Both studies were included in the tungsten trioxide submission. An adaptation for Annex X, Section8.7.3.; test method: EU B.56./OECD TG 443– Cohort 3 is been requested based on these two studies.Fastje et al. (2012) and Osterburg et al. (2014) studies were submitted in the tungsten trioxide registration. At this time, we are presenting more details on the study design and results to support our recommendation that a Cohort 3 is not justified.To determine if sodium tungstate can cause an unnatural immune response, mice were exposed in utero by parental inhalation or ingestion of an estimated dose of 1 and 4 mg/kg bw/day, respectively; and inoculated with respiratory syncytial virus (RSV) (within 2 weeks of weaning). The dams were exposed to sodium tungstate through water (15 ppm,ad libetum) and aerosol. During the 45-min, 5 days/week aerosol exposures, female mice were exposed to a 187 g/L solution nose-only for 1 week prior to conception and 3 weeks of gestation until parturition halted exposures.Pups were weaned on to tungstate-spiked water (15 ppm,ad libetum). At 21–35 days of age the mouse pups were lightly anesthetized and the nasal cavity inoculated with human RSV.Peripheral hematology was evaluated utilizing complete blood counts.Spleen tissue was massed and splenic ratio calculated as spleen mass per body mass. Results showed that controls and tungstate only-treated mice did not exhibit pathological indicators. RSV inoculation within 2 weeks of weaning was associated with a neutrophil shift. When the RSV inoculation was combined with exposure to tungstate(Na2WO4+ RSV), significant splenomegaly resulted in addition to other hematological pathologies which were not significant Exposure to Na2WO4+ RSV resulted in hematological/immunological disease, the nature of which wasinconclusive (Fastje et al. 2012). Osterburg et al. (2014) tested if exposure to sodium tungstate can result in an immune effect in a one-generation (one-gen) model and intraperitoneally injected with Staphylococcal enterotoxin (SEB). For this, parental male and female mice were orally exposed (via drinking water) to 0, 2, 62.5, 125, 200 mg sodium tungstate/kg bw/day.Both P and F1 micewere maintained on these doses for the course of the study. These tungstatedoseswere selected based onprevious workbyMcInturf et al.(2011) that used similar doses in rats. Mice were exposed to tungstate for 90-days prior to mating (Weeks 1–12). The next 7 weeks comprised gestation and weaning (Weeks 13–19). After pups (F1)were weaned, the parents (P) were necropsied, approximately19 weeks after initiation of the study. The F1 generation was exposed to tungstate for a further 90-days after weaning and then necropsied. Mice were housed singly during the course of thestudy and pair mated for breeding. After confirmation ofpregnancy, males were removed. During all phases of the one-gen study mice were kept on the appropriate tungstate dose (Osterburg et al. 2014). Results showed no statistically significant changes in bodyweight due to any tungstate dose levels.The 200 mg/kg bw/day males in the P generation show a consistenttrend towards decreased weights(Osterburg et al. 2014). Complete blood counts and hematological parameters from theblood of animals at necropsywere performed. With two exceptions (monocyte%and red blood cell distribution width), there were no statisticaldifferences in the data between P and F1(Figure 2). No significant changes in any of the parameters measured in response to tungstate, except for the percent monocytes. There were fewer lymphocytes in the F1 generation compared to the P generation (p<0.023), but this was not dose-related. Additionally, the red blood cell distribution width (RDW) was higher in the P generation vs the F1 pups(p<0.004). The percentage of monocytes was dose-dependently lower at higher concentrations when compared to control (p<0.003). Other parameters suggest a dose-dependent trend (e.g. hematocrit); however, these trends were not statistically significant (Osterburg et al. 2014). Tungstate-dependent changes were only observed in the spleens of animals. Furthermore, any statistically significant differences between the innate or immune responses of P and F1 mice were not noted. One-gen tungstate exposures resulted in reduced quantities of CD71+TH cells in theP and F1 mice for the 200 mg/kg/day dose group compared to the control groups. No statistically significant differences were noted in the overall quantity of CD3+CD4+TH cells. Were no statistically significant differences in quantities of CD3+CD8+cells. The cytotoxicCD3+CD8+CD71+cells in the P and F1 mice were decreased inSEB-challenged groups in 200 mg/kg/day group (Osterburg et al. 2014). Among cytokines measured in plasma, the only significant change was a dose-dependent quantitative decrease in interferon IFNγ levels in SEB-treated mice. Although not statistically significant, the F1 mice had an overall reduced IFNγ response, especially at the 62.5 mg/kg/day dose, compared to their parents (Osterburg et al. 2014). Overall, an immune response was confirmed by two separate in utero exposure studies when tungstate is co-exposed with an immune-stimulating agent such as RSV (Fastje et al. 2012) or SEB (Osterburg et al. 2014). When tungstate was co-exposed with an immune-stimulating agent an enlarge spleen or immunosuppression can be observed. This modulation of the normal cell-mediated immune response was also confirmed on two 28-day oral adult mice drinking water studies at similar sodium tungstate doses which co-exposed tungstate with SEB (Osterburg et al. 2014) or to P815 mastocytoma cellsex vivo, prior to functional assessment in the effector phase (Frawley et al. 2016; US NTP 2012). ECHA’s draft response also indicates a concern on delayed-type hypersensitivity (DTH) response in developing animals based on an adult study (Osterburg et al. 2014). However, on a separate study (Frawley et al. 2016; US NTP 2012) the DHT response is not observed making this effect equivocal. The available DHT studies are discussed below. Osterburg et al. (2014) exposed mice to tungstate orally at doses (0, 0.2, 2, 20, 200 mg/kg/day) in their drinking water for 28-days. Animals were sensitized to the chemical 4-Hydroxy-3-nitrophenyl acetic acid active ester (NP-O-Su) by subcutaneous injection. During the sensitization phase, appropriate tungstate doses continued to be administered to the treated mice.Ten days later, the mice were challenge into the right hind foot pad and the extent of footpad swelling was measured 24 -h post-injection. Results showed that the 200 mg/kgbw/day group resulted in significantly less swelling in the NP-O-Su challenged footpads. In the first experiment (DTH 1), both the 20 and 200 mg/kgbw/day dosed hosts had less edema than the saline. In a subsequent series (DTH 2), it was again found that there was significantly reduced swelling because of the 200 mg tungstate/kgbw/day treatment. However, at two lower doses (2 and 0.2 mg/kgbw/day), significantly reduced swelling comparedto control mice that had not been exposed to tungstate was not observed. In the US NTP immunotoxity study no effects were observed on the in vivo DTH response to Candida albicans (Frawley et al. 2016; US NTP 2012). Briefly, female mice were exposed to sodium tungstate via the drinking water at 125,250, 500, 1000, or 2000 mg/L (estimated oral doses of 30, 60, 120, 240, and 480 mg/kg bw/day) for 28-days. Mice were immunized with C. albicans by subcutaneous injection on Day 21 and challenged on Day 29 with the C.albicans antigen chitosan in the right footpad. Footpad swelling measured prior to challenge and 24-h post-challenge. Footpad swelling was calculated as [(post-measurement – premeasurement)100] and reported in terms of mm 100. A challenge only group, which received the chitosan injection on Day 29 without prior immunization with C. albicans, was included to control for non-specific footpad swelling. Results showed that no effects were observed on the delayed-type hypersensitivity response to C. albicans (Frawley et al. 2016; US NTP 2012).

Conclusion: There is sufficient (developmental) immunotoxicity and on adult mice immune system evidence that demonstrate that tungstate (the tungsten ion bioavailable at physiological conditions) causes at 200 mg/kgbw/day a modulation of the normal cell-mediated immune response only when tungstate is co-exposed with an immune-stimulating agent (such as RSV, SEB, or P815 mastocytoma cells). In addition, a concern of effects on the DHT of developing animals is based on equivocal adult immunotoxicity studies. Based on the weight of evidence, the addition of Cohort 3 in a potential EOGRT study is not justified.

Justification for classification or non-classification

No immunotoxicity studies are available for tungsten trioxide. However, data were available on sodium tungstate, which were used for read-across.sodium tungstate did not affect innate immunity, humoral immunity, or on developing hematopoietic cells in the bone marrow, and on unstimulated splenocyte phenotypes in female B6C3F1/N mice exposed via the drinking water. In addition, sodium tungstate did not adversely affect immune-organs of mice (thymus, spleen, mesenteric lymph node, popliteal lymph node, mucosa-associated lymphoid tissues, or bone marrow) and rats (McInturf et al, 2011). As potential cell-mediated immune effects are only observed under conditions of co-exposure to an immune-stimulating agent rather than direct action.Therefore, tungsten trioxide is not considered a direct immunotoxicant, and based on the lack of direct immunotoxicity effects observed, no classification is warranted.