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Immunotoxicity

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Description of key information

No immunotoxicity data of sufficient quality are available for tungsten blue oxide (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 toCandida albicansor 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 toC. 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

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

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 blue oxide (target substance).Bioaccessibility studies(IITRI 2010), indicate that the bioavailability of tungsten blue oxide via the oral route is limited when compared to sodium tungstate. Therefore, the read-across from sodium tungstate (source substance) to tungsten blue oxide (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 adaptio nforAnnex 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 theP 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 thein 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 blue oxide. 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 blue oxide is not considered a direct immunotoxicant, andbased on the lack of direct immunotoxicity effects observed, no classification is warranted.