Tungsten dioxide

Toxicity to reproduction

Administrative data

Endpoint:

developmental immunotoxicity

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

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 Dioxide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 3 in CSR
4. DATA MATRIX: See Annex 3 in CSR

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2014

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

GLP compliance:

yes

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:

- Basis for dose level selection : Doses of tungstate in the two studies were selected based on previous work that used similar doses in rats (McInturf et al., 2011).
- Exclusion of extension of Cohort 1B
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B
- Inclusioncof developmental immunotoxicity Cohort 3
- Route of administration : Drinking water
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals [if applicable]

Test material

Test material information

Constituent 1

Reference substance name:

Disodium wolframate

EC Number:

236-743-4

EC Name:

Disodium wolframate

Cas Number:

13472-45-2

Molecular formula:

Na2O4W

IUPAC Name:

Disodium dioxido(dioxo)tungsten

Test material form:

solid: crystalline

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sigma, St. Louis, MO

Test animals

Species:

mouse

Strain:

C57BL

Remarks:

C57BL6

Details on species / strain selection:

C57BL6 Mice were obtained from Charles River Laboratories (Wilmington, MA).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Wilmington, MA)
- Age at study initiation: 8–12-week-old
- Weight at study initiation: 19–22 g
- Housing: Mice were housed singly during the course of the study and pair mated for breeding. After confirmation of pregnancy, males were removed.
- Diet (e.g. ad libitum): All animals had ad libitum access to a low molybdenum diet
- Water (e.g. ad libitum): All animals had ad libitum access to filtered water
- Acclimation period: Mice were allowed to acclimatize to the animal facility for 7 days before commencement of the experimental phase
ENVIRONMENTAL CONDITIONS
- Temperature (°C): Temperature-controlled room at 22 C.
- Photoperiod (hrs dark / hrs light): During the course of the experiment, animals had a 12 h day/night cycle.

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Water bottles were changed 2–3 times weekly, always using water from the source and a 1 M sodium tungstate stock supply

Details on mating procedure:

Mice were housed singly during the course of the study and pair mated for breeding. After confirmation of pregnancy, males were removed

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

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. Body weights were recorded weekly and quantities of tungstate adjusted appropriately in water bottles. Water consumption was determined using graduated water bottles. Measurements were made daily of water consumed by mice.

Duration of treatment / exposure:

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, and 9 weeks after initiation of the study. The F1 generation was exposed to tungstate for a further 90 days after weaning and then necropsied. During all phases of the onegen study mice were kept on the appropriate tungstate dose.

Frequency of treatment:

Daily via drinking water.

Details on study schedule:

- Parental (P0) mice exposed for 12 weeks before mating, followed by gestation, birth, and weaning.
- F1 mice (offspring) exposed after weaning for 12 weeks.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

2 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

125 mg/kg bw/day (nominal)

Doses / concentrations 5

Dose / conc.:

200 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

There were eight mice per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Selected based on previous work that used similar doses in rats (McInturf et al., 2011).

- Rationale for animal assignment (if not random):
- Fasting period before blood sampling for clinical biochemistry:
- Other:

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were weekly monitored during the course of exposure.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Measurements were made daily of water consumed by mice.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: number of live, births, litter size, or sex ratio.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: The focus of these studies was to determine changes in immunological populations. Therefore, we performed complete blood counts and measured hematological parameters from the blood of animals at necropsy. With two exceptions (monocyte%
and red blood cell distribution width):
- Immune activation with Staphylococcal enterotoxin B (SEB) to induce immune response) and necropsy (spleen & blood)
- Flow cytometry: FSC vs SSC
- ELISA for or IL-6, TNFa, IL-10, and IFNg.

Statistics:

All statistical tests were performed with Systat (v11 or v13, Systat Software Inc., Chicago, IL). Statistical significance was assumed at p50.05. Comparisons between treatments (tungstate dose, immune challenge) were performed as two-way analysis of variance (ANOVA). Differences in weights were determined by repeated measures analysis of variance (RM-ANOVA). When assumptions of normality and/or equal variances were violated, non-parametric Kruskal-Wallis tests were performed. Specific post-hoc tests were performed and multiple test corrections performed when appropriate.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

no effects observed

Description (incidence and severity):

We found no statistically significant changes in body weight due to any tungstate dose levels. 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:

no effects observed

Description (incidence and severity):

Other parameters suggest a dose-dependent trend (e.g. hematocrit); however, these trends were not statistically significant. There were no significant alterations in overall quantities of hematological populations.

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

not specified

Reproductive function / performance (P0)

Reproductive performance:

no effects observed

Description (incidence and severity):

No statistically significant changes were observed in the number of live births, litter size, or sex ratio at any dose of tungstate tested.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

2

Key result

Dose descriptor:

NOEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

3

Key result

Dose descriptor:

NOEL

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: immunotoxicity

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Mortality / viability:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

The red blood cell distribution width (RDW) was higher in the P generation vs the F1 pups (p<0.004).

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

effects observed, treatment-related

Description (incidence and severity):

FLOW CYTOMETRY:
With two exceptions (monocyte% and red blood cell distribution width), there were no statistical differences in the data between P and F1. There were 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. Furthermore, any statistically significant differences between the innate or immune responses of P and F1 mice were not noted.

The percentage of monocytes was dose-dependently lower at higher concentrations when compared to control (p<0.003).
Tungstate-dependent changes in the 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.
The F1 controls in the SEB-treated groups were 7.20±0.76% and 2.85±0.53% for the 200 mg/kg/day tungstate groups (p<0.001). 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.
There were no statistically significant differences in quantities of CD3+ CD8+ cells in the one-gen study. The F1 mice CD3+ CD8+ CD71+ were 6.33±0.49% for the controls and 2.52±0.25% in the 200 mg/kg/day tungstate group (p<0.001).

CYTOKINES
Although not statistically significant, the F1 mice had an overall reduced IFN-g response, especially at the 62.5 mg/kg/day dose, compared to their parents.

Effect levels (F1)

Key result

Dose descriptor:

NOEL

Generation:

F1 (cohort 3)

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

developmental immunotoxicity

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

- Reduction in the quantity of CD71+ helper and cytotoxic T-cells present in the high-dose tungstate exposure groups (200 mg/kg/day) when challenged with SEB.
- A consistent reduced level of CD71 on SEB-treated cells in the 200 mg/kg/day tungstate group was noted. Because CD71 reductions due to tungstate were only seen in SEB-challenged mice, this would suggest that reduced CD71 levels of TH and TCTL cells in the spleens of SEB-treated mice was a result of either suppressed cell cycling or, potentially, alterations to apoptosis of these cells.
- Tungstate exposure could result in suppression of adaptive immunity. 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 (Staphylococcal enterotoxin B, SEB).