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Immunotoxicity

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Description of key information

No immunotoxicity data of sufficient quality are available for tungsten carbide (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 category approach on Annex 3 of the CSR.

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 (US 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 the in vivo delayed-type hypersensitivity (DTH) 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.

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.

C57BL6 mice were administered 0, 62.5, 125, or 200 mg/kg/day of tungstate for 28-days. There were no statistically significant changes in body weight due to any tungstate dose levels. No significant changes in water consumption due to the quantity of tungstate present in the water. Tungstate exposure for 28 days did not result in significant changes in the percentage of helper T cells (TH; CD3+ CD4+). 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. After these mice were intraperitoneally injected with Staphylococcal enterotoxin B (SEB) (20 mg/mouse) presented statistically significant reductions in the quantities of activated cytotoxic T-cells (TCTL; CD3+ CD8+ CD71+) and helper T-cells (TH; CD3+ CD4+ CD71+ ) compared to saline treated control animals. The 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 such as SEB.

Delayed Type Hypersensitivity (DHT)

ECHA’s draft response also indicates a concern on 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-nitrophenylacetic 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/kg bw/day group resulted in significantly less swelling in the NP-O-Su challenged footpads (Osterburg et al, 2014). In the first experiment (DTH 1), both the 20 and 200 mg/kg bw/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/kg bw/day treatment. However, at two lower doses (2 and 0.2 mg/kg bw/day), significantly reduced swelling compared to control mice that had not been exposed to tungstate was not observed.

In the US NTP immunotoxicity 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 DTH response to C. albicans (Frawley et al, 2016; US NTP, 2012).

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

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 [eg 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.

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 cells ex vivo, prior to functional assessment in the effector phase (Frawley et al, 2016; US NTP, 2012).

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: 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:
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:
1. HYPOTHESIS FOR THE CATEGORY 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 carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
assessment report
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 or test system and environmental conditions:
C57BL6 (28-day study male 8-12-week-old, 19-22 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.
Frequency of treatment:
Daily
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
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):
During the course of the 28-day exposures the body weights were measured to determine if there were tungstate dependent changes. There were no statistically significant changes in body weight due to any tungstate dose levels.
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
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
effects observed, treatment-related
Description (incidence and severity):
- Some sex-dependent differences in innate immune response were observed (neutrophils [Gr1++ CD11b+ ] and monocytes [Gr1-CD11b+] were different between males and females). However, these were un-related to tungstate exposure. Tungstate-dependent changes were only observed in the spleens of animals.
- In a 28-day statistically significant reductions were observed in the quantities of activated cytotoxic T-cells (TCTL; CD3+ CD8+ CD71+) and helper
T-cells (TH; CD3+ CD4+ CD71+). CD71+ TCTL cells were 12.87 2.05% (SE) in the 0 tungstate (control) group compared to 4.44 1.42% in the 200 mg/kg/day (p<0.001) group. THcells were 4.85 1.23% in controls and 2.76 0.51% in the 200 mg/kg/day (p<0.003) group.
- Tungstate exposure for 28 days did not result in significant changes in the percentage of helper Tcells (TH; CD3+ CD4+). 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 (4.85 1.23% for control vs 2.76 0.51% in tungstate group, p<0.003).
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Cell viabilities:
not specified
Humoral immunity examinations:
no effects observed
Description (incidence and severity):
No effect of the 28-day tungstate treatment on the quantity of cytotoxic T-cells (CD3+ CD8+) in the saline controls or in the SEB-challenged mice. The number of CD71+ CD8 cytotoxic T-cells was significantly reduced in the SEB-treated mice at high tungstate doses. The 0 mg/kg/day dose resulted in 12.87 2.05% CD71þ cytotoxic T-cells vs 4.44 1.42% in the 200 mg/kg/day group (p<0.001).
Other functional activity assays:
effects observed, treatment-related
Description (incidence and severity):
Among cytokines measured in plasma, the only significant change across both studies was a dose-dependent quantitative decrease in interferon (IFN)-g levels in SEB-treated mice. In the 28-day study there was a decrease from 6.98 0.77 pg/ml in the control mice to near baseline levels in the high-tungstate dose hosts (p<0.001)
Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
immunology

Taken together, the data from the current studies clearly indicate tungstate exposure could result in suppression of adaptive immunity. The 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 (eg SEB). This may suggest that some biological processes, such as T-cell activation, are more sensitive to tungstate.

Conclusions:
C57BL6 mice were administered 0, 62.5, 125, or 200 mg/kg/day of tungstate for 28-days. There were no statistically significant changes in body weight due to any tungstate dose levels. No significant changes in water consumption due to the quantity of tungstate present in the water. Tungstate exposure for 28 days did not result in significant changes in the percentage of helper T cells (TH; CD3+ CD4+). 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. After these mice were intraperitoneally injected with Staphylococcal enterotoxin B (SEB) (20 mg/mouse) presented statistically significant reductions in the quantities of activated cytotoxic T-cells (TCTL; CD3+ CD8+ CD71+) and helper T-cells (TH; CD3+ CD4+ CD71+ ) compared to saline treated control animals. The 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 such as SEB.
Executive summary:

No immunotoxicity data of sufficient quality are available for tungsten carbide (target substance). However, immunotoxicity data are available for sodium tungstate (source substance), which are 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 read-across category approach included in the Category section of this IUCLID submission on Annex 3 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:
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:
1. HYPOTHESIS FOR THE CATEGORY 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 carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
assessment report
Qualifier:
equivalent or similar to guideline
Guideline:
other:
Version / remarks:
Experiments were conducted as described in Current Protocols in Immunology
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 or test system and environmental conditions:
C57BL6 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:
During the sensitization phase, appropriate tungstate doses continued to be administered to the treated mice. Ten days later, the mice were anesthetized with isofluorane (3.5%) for injection of 20 ml of NP-O-Su mixed (1:20) with phosphate-buffered saline (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.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
C57BL6 mice were given tungstate orally by including appropriate doses (0, 0.2, 2, 20, 200 mg/kg/day) in their drinking water for 28 days.
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.:
20 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
Details on study design:
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.
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.
Specific cell-mediated immunity:
DELAYED-TYPE HYPERSENSITIVITY (DTH) REACTION:
Tungstate-dependent immune suppression in a delayed-type hypersensitivity model. Mice were exposed to tungstate in their drinking water for 28 days prior to the initiation of primary and secondary immune responses with NP-O-Su. The left footpad received saline injection, and the right footpad immunogen. Twenty-four hours after secondary challenge, footpad thickness was measured using dial gauges. The difference between left and right footpad thickness for each mouse was calculated and plotted

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 delayed-Type IV hypersensitivity experiments.
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:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
effects observed, treatment-related
Description (incidence and severity):
- 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.
Specific cell-mediated immunity:
effects observed, treatment-related
Description (incidence and severity):
In separate DTH trials (Figure 7) the 200 mg/kg/day dose resulted in significantly less swelling in the NP-O-Su challenged footpads. In the first experiment (DTH 1), both the 20 and 200 mg/kg/daydosed hosts had less edema than the saline controls (control vs 20 mg/kg/day [p<0.017]; control vs 200 mg/kg/day [p<0.013]). In a subsequent series (DTH 2), it was again found that there was significantly reduced swelling as a result of the 200 mg tungstate/ kg/day treatment (p<0.029). However, at two lower doses (ie 2 and 0.2 mg/kg/day), significantly reduced swelling compared to control mice that had not been exposed to tungstate was not observed.
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'
Conclusions:
It was shown in separate DTH experiments that there was significantly reduced footpad swelling in hosts that received the 20 and 200 mg/kg/day tungstate doses. Lower doses (ie 2 mg/kg/day; NOAEL) did not result in significant decreases in swelling.

Taken together, the DTH data from the current study indicate tungstate exposure could result in suppression of adaptive immunity. The 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 such as NP-O-Su (4-hydroxy-3-nitrophenylacetic acid active ester)
Executive summary:

No immunotoxicity data of sufficient quality are available for tungsten carbide (target substance). However, immunotoxicity data are available for sodium tungstate (source substance), which are 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 read-across category approach included in the Category section of this IUCLID submission on Annex 3 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:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
1. HYPOTHESIS FOR THE CATEGORY 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 carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
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 (ie 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 carbide (target substance). However, immunotoxicity data are available for sodium tungstate (source substance), which are 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 read-across category approach included in the Category section of this IUCLID submission on Annex 3 in the CSR. 

Endpoint:
immunotoxicity: sub-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:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
1. HYPOTHESIS FOR THE CATEGORY 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 carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Qualifier:
no guideline followed
Principles of method if other than guideline:
Assessment of B-cell development
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
C57BL
Sex:
male
Details on test animals or test system and environmental conditions:
Four-week-old male C57BL/6J mice were given food and water ad libitum. After 1 week of acclimation, mice were divided into four treatment groups
Route of administration:
oral: drinking water
Vehicle:
water
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Sodium tungstate was dissolved in tap water and was replaced every 2 or 3 days to limit conversion to polytungstates, with remaining water used to calculate average daily consumption
Duration of treatment / exposure:
Exposure was conducted over a period of 16 weeks
Frequency of treatment:
Daily via drinking water
Dose / conc.:
15 mg/L drinking water
Remarks:
Doses / Concentrations:
15 mg Tungsten/L
Basis:
nominal in water
The lowest concentration of 15 mg/l tungsten used in this study is approximately 20× higher than the highest private residential well water sampling reported within Churchill County
Dose / conc.:
200 mg/L drinking water
Remarks:
Doses / Concentrations:
200 mg Tungsten/L
Basis:
nominal in water
Dose / conc.:
1 000 mg/L drinking water
Remarks:
Doses / Concentrations:
1000 mg Tungsten/L
Basis:
nominal in water
No. of animals per sex per dose:
n = 5 per treatment group
Control animals:
yes, concurrent vehicle
Details on study design:
An additional study compared removal of tungsten for 4 and 8 weeks, following a 4-week exposure to 15 mg/l tungsten
Sacrifice and pathology:
- Liver toxicity was assessed in tungsten-exposed mice by measuring aspartate aminotransferase (AST) and alanine transaminase (ALT) enzyme levels in peripheral blood postmortem.
- Tibia bones were weighed and measured prior to flushing the bone marrow. Marrow was flushed from both tibiae and femora for flow cytometric analyses, colony forming unit (CFU) assays, and assessment of DNA damage
Cell viabilities:
BONE MARROW: Yes
- Method: The colony forming unit pre-B (CFU-pre-B) assay was performed on freshly isolated bone marrow to determine common lymphoid progenitor (CLP) cell proliferation and differentiation capacity within the marrow
- Dose groups: 15, 200, or 1000 mg/l tungsten
- No. of animals: 5
Other functional activity assays:
ENUMERATION TOTAL B CELLS
- Method: Flow cytometry
- Dose groups: 15, 200, or 1000 mg/l tungsten
- No. of animals: 5

Other examinations:
- Hematology, liver function, and tungsten analyses
Statistics:
One-way ANOVA followed by Newman-Keuls post hoc test was used to determine differences between data sets
Clinical signs:
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Although animal weight was significantly lower in animals given the highest concentration of tungsten compared with all other groups, these animals still appeared to gain weight at a normal rate
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):
Daily water consumption between groups did not differ (+/- 0.16 ml/g).
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
- Tungsten exposure did not alter erythrocyte and platelet counts or hemoglobin and hematocrit levels
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Results did not show liver toxicity as assessed by peripheral blood AST and ALT enzyme levels
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
effects observed, treatment-related
Description (incidence and severity):
- Peripheral white blood cell (WBC) counts were dose dependently decreased following 1 week of exposure. Differential analysis of the blood showed that the percentage of lymphocytes, monocytes, and granulocytes within the leukocyte compartment remained the same, indicating a dose-dependent panleukopenia as a result of exposure. In later weeks, the decreased WBCs were only significant at 12 weeks of exposure at the highest concentration tested although the observed trend was present following 8 weeks of exposure.
- After 16 weeks of exposure, all tungsten-exposed groups had a significantly greater percentage of cells in fraction C/C′, which constitutes the late pro- and large pre-B cell stages (Fig. 5A). The greater percentage of fraction C/C′ cells resulted in a significant increase in the total number of C/C′ cells (15 and 200 mg/l concentrations) when normalized to bone marrow cellularity.
- Total bone marrow cellularity only differed significantly after 16 weeks of treatment with 1000 mg/l tungsten


Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No significant differences were observed in either their tibia weight or length compared with the control animals
Gross pathological findings:
no effects observed
Description (incidence and severity):
No significant differences were observed in either their tibia weight or length compared with the control animals
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The amount of tungsten in the tibia bones of exposed animals was quantitated using ICP-MS over the 16-week experiment. Tungsten concentration increased rapidly following the first week of exposure, after which the accumulation rate decreased significantly (p < 0.001), for the remainder of the experiment. Tungsten content of tibiae was dependent upon the concentration of tungsten to which the individual was exposed. Interestingly, lower exposures suggest an incomplete saturation of the tibia compartment, as tungsten content here peaked at values below the bone concentration achieved with our highest exposure.
- After 4 weeks without tungsten, tibia tungsten levels decreased significantly to approximately half the levels of those animals with continuous tungsten exposure. No further decrease from this level after 8 weeks on tap water was found.
Details on results:
- Differential analysis of the blood showed that the percentage of lymphocytes, monocytes, and granulocytes within the leukocyte compartment remained the same, indicating a dose-dependent panleukopenia as a result of exposure.
- In later weeks, the decreased WBCs were only significant at 12 weeks of exposure at the highest concentration tested although the observed trend was present following 8 weeks of exposure.
- Tungsten content of tibiae was dependent upon the concentration of tungsten to which the individual was exposed.
- Flow cytometric analyses revealed a transient increase in mature IgD+ B cells in the first 8 weeks of treatment, in animals of the highest and intermediate exposure groups. Following 16 weeks of exposure, all tungsten groups had a significantly greater percentage of cells in the late pro-/large pre-B developmental stages.
- Total bone marrow cellularity only differed significantly after 16 weeks of treatment with 1000 mg/l tungsten
Cell viabilities:
no effects observed
Description (incidence and severity):
- The number of CLP (defined as Lin−, Sca-1low, c-kitlow, and IL-7R+) was assessed by flow cytometry in both control and tungsten-exposed animals, but no differences were observed
- In vivo tungsten treatment resulted in a dose-dependent increase in the number of colonies detected compared with age-matched controls following 16 weeks of exposure, and although not significant, this trend was observed in as early as 1 and 4 weeks of exposure
Humoral immunity examinations:
not specified
Specific cell-mediated immunity:
not specified
Non-specific cell-mediated immunity:
not specified
Other functional activity assays:
effects observed, treatment-related
Description (incidence and severity):
Peripheral white blood cell (WBC) counts were dose dependently decreased following 1 week of exposure
Other findings:
effects observed, treatment-related
Description (incidence and severity):
- Tungsten exposure resulted in a significant increase in comet tail moment. Intriguingly, a significant increase was observed at the lower concentration of tungsten but not at the highest concentration.
- Increased DNA damage was observed at lower tungsten concentrations of 15 and 200 mg/l but not the 1000 mg/l concentration from marrow of animals exposed for 1 or 4 weeks to tap water with and without tungsten
- Immunoblotting experiments of CD19+ B cells isolated from control and animals exposed to tungsten for 1 week showed an increase in γH2AX, particularly at the lower concentrations
Key result
Dose descriptor:
LOAEL
Effect level:
15 mg/L drinking water
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Sodium tungstate exposure results in an alteration of B-cell development profile.
Conclusions:
This study provides evidence that sodium tungstate exposure results in an alteration of B-cell development profile.
Executive summary:

No  immunotoxicity data of sufficient quality are available for tungsten carbide (target substance). However,  immunotoxicity data are available for sodium tungstate (source substance), which are 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 read-across category approach included in the Category section of this IUCLID submission on Annex 3 in the CSR. 

Endpoint:
immunotoxicity: short-term 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:
1. HYPOTHESIS FOR THE CATEGORY 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 carbide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR
Reason / purpose for cross-reference:
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 or test system 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 drinking 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 carbide (target substance). However, immunotoxicity data are available for sodium tungstate (source substance), which are 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 read-across category approach included in the Category section of this IUCLID submission on Annex 3 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.

Effect on immunotoxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Effect on immunotoxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

No immunotoxicity studies are available for tungsten carbide (the target substance). However, data were available on sodium tungstate (the source substance), 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, sodium tungstate is not considered a direct immunotoxicant, and based on the lack of direct immunotoxicity effects observed.

Furthermore, based on bioaccessibility studies of tungsten carbide that reported a 0.04% bioelution of tungsten after 5-hrs. incubation in gastric fluid, the equivalent amount of tungsten carbide to reach NOAEL doses is substantially higher than 1,000 mg/kg/day (limit dose). Therefore, no hazard classification is warranted for tungsten carbide.