Registration Dossier

Administrative data

Description of key information

Repeated Dose Toxicity - Oral Route:

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

The read across study on sodium tungstate was sponsored conducted the United States Army Center for Health Promotion and Preventive Medicine and published by McCain et al. (2015). The 90-day oral toxicity study was conducted in rats according to the procedure described in the Environmental Protection Agency (EPA) Health Effects Testing Guidelines (40 CFR, Part 798.2650) in compliance with Good Laboratory Practice. Briefly, this study of the subchronic toxicity of sodium tungstate dihydrate aqueous solution in male and female Sprague-Dawley rats was evaluated by daily oral gavage of 0, 10, 75, 125, or 200 mg/kg bw/d for 90 days. Measured parameters included food consumption, body weight measurements, hematology, clinical chemistry, and histopathological changes. There was a significant decrease in food consumption and body weight gain in males at 200 mg/kg bw/d from days 77 to 90; however, there was no effect in food consumption and body weights in females. There were no changes in the hematological and clinical parameters studied. Histopathological changes were seen in kidney of male and female and epididymis of male rats. The histopathological changes observed in the kidneys of male and female rats dosed at 125 or 200 mg/k/d consisting of mild to severe cortical tubule basophilia in 2 high-dose groups. Histological changes in epididymides included intraluminal hypospermia with cell debris in the 200 mg/kg bw/day dosed male rats. Histopathological changes were observed in the glandular stomach including inflammation and metaplasia in the high-dose groups (125 or 200 mg/kg bw/day) of both sexes of rats. Based on histopathology effects seen in the kidneys, the lowest observable adverse effect level was 125 mg/kg bw/d and the no observable adverse effect level was 75 mg/kg bw/d in both sexes of rats for oral subchronic toxicity. The USEPA’s Benchmark Dose Software (BMDS, Version 1.4.1) was used to model the data to derive a BMDL10. The lowest (most precautionary) BMDL10 from the renal toxicity endpoint in the 90-day oral toxicity study was 102 mg/kg bw/d.

In addition to McCain et al. (2015) rat oral 90-day repeated dose study, the US National Toxicology Program (NTP) has conducted two additional 90-day drinking water studies, one in Sprague-Dawley rats and a second one in B6C3F1 mice (10/sex/species/dose). The study design included doses of0, 125, 250, 500, 1000, or 2000 mg/L. The in-life study phase has been completed but no study report has yet been issued. Currently, available in the US NTP website are graphs and Tables are preliminary results, but no full report has been issued. Furthermore, at the 2012 Annual Meeting of the Society of Toxicology, a Scientific Poster was presented detailing preliminary results of the NTP study. Preliminary results confirm the results of the McCain gavage study, showing the kidney as the major target organ for tungstate (especially at high drinking water doses of 1,000 and 2,000 mg/L). In a personal communication, the U.S. NTP Study Coordinator, Dr. Mamta Behl estimated that final reports will be available in 2 to 3 years.

Repeated Dose Toxicity - Inhalation Route:

No inhalation repeated dose toxicity data of sufficient quality were available for tungsten trioxide; however, data were available for tungsten oxide, which will be used for reading-across A 28-day inhalation toxicity study conducted according to OECD 412 is available on tungsten blue oxide, which is considered the key repeated dose study. In this study, 5 rats/sex/dose were given TBO nose-only for 6 hours per day, 7 days/week, for 28 days (with a 14-day recovery period) at doses of 0 (control), 0.08, 0.325, and 0.65 mg/L air. The NOAEC was deemed to be > 0.65 mg/L air (650 mg/m3), as no significant effects were reported.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: 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
Remarks:
Well documented, scientifically sound study that was conducted according to EPA OTS 798.2650 (90-Day Oral Toxicity in Rodents)
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium tungstate
Target: Tungsten Trioxide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Reason / purpose:
assessment report
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
EPA OTS 798.2650 (90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Sodium tungstate dihydrate (Tungstic acid sodium salt dihydrate, Na2WO42H2O, CAS # 10213-10-2, Batch # 12330JO) 99% pure
Species:
rat
Strain:
other: SD
Details on species / strain selection:
5-week-old SD rats were purchased from Charles River laboratories, Raleigh, North Carolina
Sex:
male/female
Details on test animals and environmental conditions:
Animals were held for 1 week in quarantine prior to initiation of treatments. At the start of testing, rats weighed between 199 and 230 g. Test animals were identified by individual cage cards and microchip implants and were individually housed in
polycarbonate cages. Bedding was placed in the bottom of each cage and replaced twice weekly. Drinking quality water and a certified laboratory diet were available ad libitum. Animal rooms were maintained at 64 F to 79 F, with relative humidity of 30% to 70% and a 12-hour light–dark cycle.
Route of administration:
oral: drinking water
Details on route of administration:
Sodium tungstate dihydrate was solubilized with DI water to produce 4 dosing solutions
Vehicle:
water
Details on oral exposure:
The dose levels were selected on the basis of our previous studies where the highest dose used was 200 mg/kg/ d in a subchronic toxicity study. Sodium tungstate dihydrate was solubilized with DI water to produce 4 dosing solutions of
200, 125, 75, and 10 mg Na2WO4/mL.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Tungstate concentrations of the dosing solutions were verified by the Aberdeen Test Center and found to be consistent for purity and stability during the study period
Duration of treatment / exposure:
A 90-day oral toxicity study
Frequency of treatment:
Test chemical solutions were administered daily (7 days per week) for 90 days. A 16 GA 2-in stainless steel gavage needle
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Following 1-week quarantine/acclimatization period, 50 male and 50 female SD rats were randomly distributed
Control animals:
yes, concurrent vehicle
Details on study design:
Sodium tungstate dihydrate was solubilized with DI water to produce 4 dosing solutions of 200, 125, 75, and 10 mg Na2WO4/mL.
Observations and examinations performed and frequency:
A clinical examination was made for each animal prior to initiation of treatment and once weekly during treatment. Observations included but were not limited to changes in skin and fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (eg, lacrimation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture, and response to handling as well as the presence of clonic or tonic movements, stereotypes (eg, excessive grooming, repetitive circling), or bizarre behavior (eg, selfmutilation, walking backward) were recorded according to testing guidelines. Body and feeder weights were recorded on days .3, .1, 0 (first day of dosing), 7, and weekly thereafter. Doses were adjusted weekly to reflect the change in individual body weights. Animals were observed daily for any toxic signs.
Sacrifice and pathology:
Blood was collected. Each rat was then submitted for complete necropsy. The brain, heart, liver, kidneys, spleen, adrenals, thymus, epididymis/uterus, and testes/ovaries were removed and weighed for absolute organ weights. The tissues harvested for histopathological evaluation included the brain, pituitary, thyroid with parathyroid gland, thymus, lungs, trachea, heart, bone marrow, salivary gland, liver, spleen, kidney, adrenal gland, pancreas, testis, ovaries, uterus, aorta, esophagus, stomach, duodenum, jejunum, ileum, caecum, colon, urinary bladder, salivary lymph node peripheral nerve (siatic), thigh musculature (vastus lateralis), eye, spinal cord (3 levels), and exorbital lachrymal gland.

Hematology parameters included white blood cell count, neutrophils, lymphocytes, monocytes, eosinophils, basophils, red blood cell count, hemoglobin, hematocrit, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, red blood cell distribution width, platelets, and mean platelet volume.

Clinical chemistry parameters measured included The clinical chemistry analytes ialkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, calcium, cholesterol, creatinine kinase, creatinine, glucose (GLU; nonfasting), lactate dehydrogenase, total bilirubin, total protein, triglycerides, Na, K, and Cl

Urinalysis included appearance, pH, specific gravity, GLU, bilirubin, urobilinogen, ketone, blood, protein, nitrite, and leukocytes.
Other examinations:
Ophthalmic examinations were performed on all control and treated animals prior to the scheduled start of the study and within a week of the scheduled 90-day necropsies. Urinalysis was also performed on 8 of 10 animals from all dose
groups (including negative control) within 2 weeks of the final (90-day) necropsies
Statistics:
Food consumption, body weights, and absolute organ weights were compared among dosage groups and controls using ANOVA. When significance was observed, the data were further analyzed using a Dunnett test to compare the doses to the control group. Statistical significance was defined at the P <05 level. Clinical chemistry, hematology, and urinalysis data were analyzed with ANOVA and Bonferroni post hoc test to compare dosage groups to the control group.
Clinical signs:
no effects observed
Description (incidence and severity):
- No evidence of overt toxicity and no treatment-related clinical signs were seen in any dose levels.
- Results showed that rats dosed with sodium tungstate in water for 90 consecutive days had no abnormal clinical signs at any of the dose levels
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No unscheduled deaths (a single male rat in 200 mg/kg/d dose group was moribund and was euthanized on day 79; a few tissues from this rat were submitted for histopathological examination and death was determined to be not compound related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were significant decreases in food consumption in male rats at 200 mg/kg/d from weeks of 4 to 13, while there was no change in food consumption in female rats during the 90-day study
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There were significant decreases in food consumption in male rats at 200 mg/kg/d from weeks of 4 to 13, while there was no change in food consumption in female rats during the 90-day study changes in female rats in any dose groups throughout study
period when compared to control rats.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmic examinations prior to study initiation and within
a week of the scheduled necropsies revealed no abnormalities
Haematological findings:
no effects observed
Description (incidence and severity):
Male and female rats showed no significant differences in any hematological parameters at any dose levels of sodium tungstate
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The results of clinical chemistry parameters studied in rats showed no significant changes in any dose levels of sodium tungstate in rats. The parameters studied showed some changes in levels that were not dose related and insignificant and considered within normal range limits when compared to controls. All other parameters were found to be similar to control rats.

Urinalysis findings:
no effects observed
Description (incidence and severity):
Examination of urine samples taken approximately 1 week prior to necropsy revealed no significant changes in volume, specific gravity, or pH. No distinct dose-related trends were observed in GLU, bilirubin, ketone, blood, protein, urobilinogen,
nitrite, or leukocytes
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The body weights, absolute heart, liver, and thymus weights were significantly lower in male rats dosed at 200 mg/kg/d compared to control rat, but there were no effects on body weights and organ weights of female rats
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Histopathology examination revealed effects on the urogenital system of the sodium tungstate-treated rats. Changes included mild to severe basophilia of renal cortical tubules in 1 of 9 and 10 of 10 males and 1 of 10 and 8 of 10 females in 2 high-dose
groups (125 and 200 mg/kg/d), respectively.
- Histopathological analysis of epididymides of rats dosed with sodium tungstate showed considerable effects in the high-dose group, Intraluminal cellular debris with and without hypospermia was noted in the epididymides of 3 of 10 males in the 200 mg/kg/d dose group. The lesion was not observed in the 10, 75, and 125 mg/kg/d dose groups.
- Histologic changes were also noted in the glandular stomach of males and females in high dosage groups. The changes included subacute inflammation consisting primarily of EOSs admixed with fewer mononuclear cells observed throughout the submucosa of 5 of 9, 4 of 10 males, and 8 of 10, 9 of 10 females in, 125 and 200 mg/kg/d dosage groups, respectively. Goblet cell metaplasia was also observed in the mucosa of the glandular stomach 8 of 9, 8 of 10 males and 8 of 10, 10 of 10
females of 125 and 200 mg/kg/d dosage groups, respectively. The gastric histologic findings in the lower dosed group were negative when compared to 2 high-dose groups
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
BMDL10
Effect level:
102 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
LOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
LOAEL
Effect level:
175 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
LOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
food consumption and compound intake
Key result
Dose descriptor:
LOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
haematology
Key result
Dose descriptor:
LOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
125 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Conclusions:
Subchronic toxicity of sodium tungstate was assessed in in male and female Sprague-Dawley rats by daily oral gavage of 0, 10, 75, 125, or 200 mg/kg/d for 90 days. There was a significant decrease in food consumption and body weight gain in males at 200 mg/kg/d from days 77 to 90; however, there was no effect in food consumption and body weights in females. There were no changes in the hematological and clinical parameters studied. Histopathological changes were seen in kidney of male and female and epididymis of male rats. Histopathological changes were observed in the kidneys of male and female rats dosed at 125 or 200 mg/k/d consisting of mild to severe cortical tubule basophilia in 2 high-dose groups. Histological changes in epididymides included intraluminal hypospermia with cell debris in the 200 mg/kg/d dosed male rats. Histopathological changes were observed in the glandular stomach including inflammation and metaplasia in the high-dose groups (125 or 200 mg/kg/d) of both sexes of rats. Based on histopathology effects seen in the kidneys, the lowest observable adverse effect level was 125 mg/kg/d and the no observable adverse effect level was 75 mg/kg/d in both sexes of rats for oral subchronic toxicity.
Executive summary:

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

Endpoint:
sub-chronic toxicity: oral
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:
comparable to guideline study with acceptable restrictions
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium tungstate
Target: Tungsten Trioxide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Details on species / strain selection:
B6C3F1/N
Sex:
male/female
Details on test animals and environmental conditions:
After a 10- to 14-day quarantine period, animals are assigned at random to treatment groups including:
- Five treatment groups, each administered a different concentration of the test substance
- One control group

Each group contains 10 animals per sex per species. Male mice are housed individually,

Animals are individually weighed on days one and seven, and at sacrifice. All animals are observed twice daily for clinical signs of pharmacologic and toxic effects of the test substance, declining health, or death. Animals found near death or showing clinical signs of pain or distress are humanely euthanized. For dosed-feed or dosed-water studies, food consumption/water consumption is measured and recorded weekly.
Route of administration:
oral: drinking water
Details on route of administration:
doionized drinking water
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Frequency of treatment:
Daily
Dose / conc.:
0 mg/L drinking water
Dose / conc.:
125 mg/L drinking water
Dose / conc.:
250 mg/L drinking water
Dose / conc.:
500 mg/L drinking water
Dose / conc.:
1 000 mg/L drinking water
Dose / conc.:
2 000 mg/L drinking water
No. of animals per sex per dose:
Each group per sex per species contains five animals
Control animals:
yes, concurrent vehicle
Positive control:
Not applicable
Observations and examinations performed and frequency:
Animals are individually weighed on days one, seven, and at weekly periods thereafter. All animals are observed twice daily for clinical signs of declining health, or death. Animals found near death or showing clinical signs of pain or distress are humanely euthanized. Formal clinical observations are performed and recorded weekly. Food consumption/water consumption is measured and recorded weekly.

Clinical Laboratory Studies
Blood is collected from both sexes of "special study" rats, at days 4 ± 1 and 21 ± 2 and from the core study rats at the end of the study. These are processed for hematology and clinical chemistry determinations. Blood is collected from core study mice at the end of the study for hematology determinations. See clinical measurements:

1. Hematology:
Erythrocyte count
Mean corpuscular volume
Hemoglobin
Packed cell volume
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Erythrocyte morphologic assessment
Leukocyte count
Leukocyte differential
Reticulocyte count
Platelet count and morphologic assessment

2. Clinical Chemistry:
Sorbitol dehydrogenase (SDH)
Alkaline Phosphatase (ALP)
Creatine Kinase (CK)
Creatinine
Total Protein
Albumin
Urea Nitrogen (BUN)
Total Bile Acids
Alanine Aminotransferase (ALT)
Glucose
Cholesterol
Triglycerides
Sacrifice and pathology:
A complete gross necropsy is an external examination of the animal including body orifices and examination and fixation of all of the following organs/tissues from animals from all treatment groups for histopathologic examination.
Adrenal glands
Brain
Clitoral glands
Esophagus
Eyes
Femur
Gallbladder (mouse)
Gross lesions
Harderian glands
Heart and aorta
Intestine, large (cecum, colon, rectum)
Intestine, small (duodenum, jejunum, ileum)
Kidneys
Liver
Lungs and mainstem bronchi
mandibular and mesenteric
bronchial mediastinal (inhalation studies)
Mammary gland with adjacent skin
Muscle, thigh
Nerve, sciatic
Nasal cavity and nasal turbinates
Oral cavity, larynx, and pharynx
Ovaries
Pancreas
Parathyroid glands
Pituitary gland
Preputial glands
Prostate
Salivary glands
Seminal vesicles
Skin, site of application (dermal studies)
Spinal cord
Spleen
Stomach (forestomach and glandular)
Testes, epididymides, and vaginal tunics of testes
Thymus
Thyroid gland
Tissue masses
Tongue
Trachea
Urinary bladder
Uterus
Vagina
Zymbal glands

- A complete histopathologic evaluation inclusive of treatment-related gross lesions shall be done on all animals. Treatment-related lesions for target organs shall be identified and these organs plus gross lesions shall be examined to a no-effect level. TIssues examined:
Adrenal glands
Brain (3 sections including frontal cortex and basal ganglia, parietal cortex and thalamus, and cerebellum and pons)
Clitoral glands
Esophagus
Eyes
Femur, including diaphysis with marrow cavity and epiphysis (femoral condyle with epiphyseal cartilage plate, articular cartilage and articular surface)
Gallbladder (mouse)
Gross lesions
Harderian glands
Heart and aorta
Intestine, large (cecum, colon, rectum)
Intestine, small (duodenum, jejunum, ileum)
Kidneys
Larynx (inhalation studies)
Liver (2 sections including left lateral lobe and median lobe)
Lungs and mainstem bronchi
Lymph nodes
mandibular and mesenteric
bronchial & mediastinal (inhalation studies)
Mammary gland with adjacent skin
Muscle, thigh (only if neuromuscular signs were present)
Nasal cavity and nasal turbinates (3 sections)
Ovaries
Pancreas
Parathyroid glands
Pituitary gland
Preputial glands
Prostate
Salivary glands
Seminal vesicle
Skin, site of application (dermal studies)
Spinal cord and sciatic nerve (if neurologic signs were present)
Spleen
Stomach (forestomach and glandular)
Testes with epididymides
Thymus
Thyroid gland
Tissue masses
Trachea
Urinary bladder
Uterus
Other examinations:
Genotoxicity (micronucleus and Comet assay)
Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
During the 13-week phase of the study, there was no effect on survival in mice
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Male mice at the highest drinking water concentration (2000 mg/L) showed a decreased on body weight starting at the 15-day of treatment compared it to control animals. Whereas female mice body weight started to decreased at the fourth week of treatment at all the drinking water concentrations.
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, treatment-related
Description (incidence and severity):
Decreased water consumption was observed in 1000 (11%) and 2000 mg/L (16%) male mice
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
During the 13-week phase of the study, there was no effect on hematology in mice
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
During the 13-week phase of the study, there was no effect on organ weights in mice
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Liver cellular infiltration (mixed cell) was found in 30 and 40% of the male mice in control and 2000 mg/L animals, respectively.
- Cellular infiltration (mononuclear cell) was observed in 10% of the male animals in control, 125 and 1000 mg/L treatment groups. Twenty percent of the male animals at the 2000 mg/L presented also cellular infilration. Nephropathy was found in control (10%), 125 (10%), 250 (10%), 500 (10%) and 1000 (20%) mg/L groups. Renal tubule regeneration was reported in 60 and 100% of the animals exposed to 1000 and 2000 mg/L, respectively.
- Ten percent of the female control animals presented inflammation of the large (rectum) and small (jejunum) intestines, and salivary gland cellular infiltration; with 90% of the control female mice presented liver cellular infiltration (mixed cell).
- Ninety percent of the 2000 mg/L female mice presented liver cellular infiltration (mixed cell), with 10% of the female mice perivascular lung cellular infiltration (mononuclear), bone lession (fibro-osseous), and and salivary gland cellular infiltration.
- Nephropathy was reported in 10 and 20% of the female mice in the 125 and 250 mg/L groups. Kidney cellular infiltration (mononuclear) was observed in 10% of the female mice in the 500 and 1000 mg/L groups. Renal tubule (regeneration) was reported in 10 and 20% of the female mice in the 1000 and 2000 mg/L.
- Statistically significance of non-neoplastic lessions (kidney renal tubule regeneration) in male mice were reported at 1000 and 2000 mg/L drinking water concentration
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
- No neoplasms were reported in exposed male or female mice.
Other effects:
no effects observed
Description (incidence and severity):
The micronucleus assay was negative in mice. The Comet assay was positive in the liver and ileum of male mice and negative in the blood and kidney of mice.
Key result
Dose descriptor:
LOAEL
Effect level:
ca. 1 000 mg/L drinking water
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 500 mg/L drinking water
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/L drinking water
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Conclusions:
Decreased water consumption was observed in 1000 (11%) and 2000 mg/L (16%) male mice. During the 13-week phase of the study, there was no effect on survival, hematology, or organ weights in mice. Renal tubule regeneration was characterized by hyperplasia of tubular epithelial cells with cytoplasmic basophilia, nuclear crowding, karyomegaly, and occasional mitotic figures. Total tungsten concentrations were generally dose proportional in blood and urine. The micronucleus assay was negative in mice. The Comet assay was positive in the liver and ileum of male mice and negative in the blood and kidney of mice. The kidney appeared to be the only major target organ following exposure of mice to sodium tungstate dihydrate at water concentrations of 1000 and 2000 mg/L.
Executive summary:

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

Endpoint:
sub-chronic toxicity: oral
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:
comparable to guideline study with acceptable restrictions
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium tungstate
Target: Tungsten Trioxide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Reason / purpose:
reference to same study
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
After a 10- to 14-day quarantine period, animals are assigned at random to treatment groups including:
- Five treatment groups, each administered a different concentration of the test substance
- One control group

Each group contains 10 animals per sex per species. Male mice are housed individually,

Animals are individually weighed on days one and seven, and at sacrifice. All animals are observed twice daily for clinical signs of pharmacologic and toxic effects of the test substance, declining health, or death. Animals found near death or showing clinical signs of pain or distress are humanely euthanized. For dosed-feed or dosed-water studies, food consumption/water consumption is measured and recorded weekly.
Route of administration:
oral: drinking water
Details on route of administration:
doionized drinking water
Vehicle:
water
Details on oral exposure:
- 90 days for dosed-feed and dosed-water studies
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90-days
Frequency of treatment:
Daily
Dose / conc.:
0 mg/L drinking water
Dose / conc.:
125 mg/L drinking water
Dose / conc.:
250 mg/L drinking water
Dose / conc.:
500 mg/L drinking water
Dose / conc.:
1 000 mg/L drinking water
Dose / conc.:
2 000 mg/L drinking water
No. of animals per sex per dose:
Each group per sex per species contains five animals
Control animals:
yes, concurrent vehicle
Positive control:
Not applicable
Observations and examinations performed and frequency:
Animals are individually weighed on days one, seven, and at weekly periods thereafter. All animals are observed twice daily for clinical signs of declining health, or death. Animals found near death or showing clinical signs of pain or distress are humanely euthanized. Formal clinical observations are performed and recorded weekly. Food consumption/water consumption is measured and recorded weekly.

Clinical Laboratory Studies
Blood is collected from both sexes of "special study" rats, at days 4 ± 1 and 21 ± 2 and from the core study rats at the end of the study. These are processed for hematology and clinical chemistry determinations. Blood is collected from core study mice at the end of the study for hematology determinations. See clinical measurements:

1. Hematology:
Erythrocyte count
Mean corpuscular volume
Hemoglobin
Packed cell volume
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Erythrocyte morphologic assessment
Leukocyte count
Leukocyte differential
Reticulocyte count
Platelet count and morphologic assessment

2. Clinical Chemistry:
Sorbitol dehydrogenase (SDH)
Alkaline Phosphatase (ALP)
Creatine Kinase (CK)
Creatinine
Total Protein
Albumin
Urea Nitrogen (BUN)
Total Bile Acids
Alanine Aminotransferase (ALT)
Glucose
Cholesterol
Triglycerides
Sacrifice and pathology:
- Liver, thymus, right kidney, right testis, heart, and lung weights are recorded from all animals surviving until the end of the study.
- A complete necropsy is performed on all treated and control animals, and all tissues required for complete histopathology are trimmed, embedded, sectioned, and stained with hematoxylin and eosin for histopathologic evaluation. See necropsy list:

A complete gross necropsy is an external examination of the animal including body orifices and examination and fixation of all of the following organs/tissues from animals from all treatment groups for histopathologic examination.
Adrenal glands
Brain
Clitoral glands
Esophagus
Eyes
Femur
Gallbladder (mouse)
Gross lesions
Harderian glands
Heart and aorta
Intestine, large (cecum, colon, rectum)
Intestine, small (duodenum, jejunum, ileum)
Kidneys
Liver
Lungs and mainstem bronchi
mandibular and mesenteric
bronchial mediastinal (inhalation studies)
Mammary gland with adjacent skin
Muscle, thigh
Nerve, sciatic
Nasal cavity and nasal turbinates
Oral cavity, larynx, and pharynx
Ovaries
Pancreas
Parathyroid glands
Pituitary gland
Preputial glands
Prostate
Salivary glands
Seminal vesicles
Skin, site of application (dermal studies)
Spinal cord
Spleen
Stomach (forestomach and glandular)
Testes, epididymides, and vaginal tunics of testes
Thymus
Thyroid gland
Tissue masses
Tongue
Trachea
Urinary bladder
Uterus
Vagina
Zymbal glands

- A complete histopathologic evaluation inclusive of treatment-related gross lesions shall be done on all animals. Treatment-related lesions for target organs shall be identified and these organs plus gross lesions shall be examined to a no-effect level. TIssues examined:
Adrenal glands
Brain (3 sections including frontal cortex and basal ganglia, parietal cortex and thalamus, and cerebellum and pons)
Clitoral glands
Esophagus
Eyes
Femur, including diaphysis with marrow cavity and epiphysis (femoral condyle with epiphyseal cartilage plate, articular cartilage and articular surface)
Gallbladder (mouse)
Gross lesions
Harderian glands
Heart and aorta
Intestine, large (cecum, colon, rectum)
Intestine, small (duodenum, jejunum, ileum)
Kidneys
Larynx (inhalation studies)
Liver (2 sections including left lateral lobe and median lobe)
Lungs and mainstem bronchi
Lymph nodes
mandibular and mesenteric
bronchial & mediastinal (inhalation studies)
Mammary gland with adjacent skin
Muscle, thigh (only if neuromuscular signs were present)
Nasal cavity and nasal turbinates (3 sections)
Ovaries
Pancreas
Parathyroid glands
Pituitary gland
Preputial glands
Prostate
Salivary glands
Seminal vesicle
Skin, site of application (dermal studies)
Spinal cord and sciatic nerve (if neurologic signs were present)
Spleen
Stomach (forestomach and glandular)
Testes with epididymides
Thymus
Thyroid gland
Tissue masses
Trachea
Urinary bladder
Uterus
Other examinations:
- Tungsten concentrations in blood and urine
- Genotoxicity (micronucleus and Comet assay): Blood for Micronuclei Blood samples are taken from mice and rats at study termination for micronuclei determinations.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Nasal/Eye discharge (at Day 35) was observed in one female of control and 250 mg/L group.Ulcer/Abscess was reported (at Day 70) in one female of the 500 mg/L group.
Mortality:
no mortality observed
Description (incidence):
All female and male rats were alve after 90-day exposure to sodium tungstate.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Bpdy weigths of male rats exposed to 000 and 2000 mg/L were lower than vehicle controls.males.
- Body weights of female rats exposed to 2000 mg/L was lower than vehicle control females.
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, treatment-related
Description (incidence and severity):
Decreased water consumption was observed in 1000 and 2000 mg/L rats
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
During the 13-week phase of the study, there was no effect on hematology
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
During the 13-week phase of the study, there was no effect on organ weights in rats
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Liver heptadiaphragmatic nodule was observed in one male of the 2000 mg/L group.
- Cellular infiltration (mixed cell) was observed in one and two male rats of the control and 2000 mg/L group, respectively.
- Preputial gland cellular infiltration (lymphocyte) was observed in 2 and 4 animals of male rats in controls and 2000 mg/L groups, respectively.
- Preputial gland inflammation and acute inflammation were observed in one control male and one male of the 2000 mg/L group, respectively. Lung infiltarion was found in one male of the control group.
- Nephropathy was reported in male rats of control (n=10), 125 (n=9), 250 (n=8), 500 (n=9), 1000 (n=8) and 2000 (n=9) mg/L. Renal tubule regenaration was reported in 3 males (30%) and 10 males (100%) of the 1000 and 2000 mg/L groups.
- One female of the 2000 mg/L presented liver hepatodiaphragmatic nodule, and liver cellular infiltration (mixed cell) was reported in 3 and 2 females of the control and 2000 mg/L groups.
- Clitoral gland cellular infiltration (lymphocyte) was observed in 1 and 2 females of the control and 2000 mg/L groups, respectively.
- Lung metaplasia (osseous) was found in one female of the control group.
- Kidney cyst (focal) was found in one female of the 2000 mg/L group, and kidney cellular infiltration (lymphocyte) was found in one female of the 125 and 500 mg/L groups. Kidney mineralization wasobserve din on single female of the 2000 mg/L group.
- Kidney nephropathy was reported in female of control (n=6), 125 (n=6), 250 (n=7), 500 (n=6), 1000 (n=5) and 2000 (n=10) mg/L groups. Kidney renal tubule regeneration was reported in females of 1000 (n=3) and 2000 (n=10) mg/L groups.

Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Neoplasms were not identified in male or female rats
Other effects:
no effects observed
Description (incidence and severity):
Total tungsten concentrations were generally dose proportional in blood and urine. The micronucleus assay was negative in rats. The Comet assay was positive in the liver of rats but negative in the blood and kidney.
Key result
Dose descriptor:
LOAEL
Effect level:
ca. 1 000 mg/L drinking water
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 500 mg/L drinking water
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/L drinking water
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Conclusions:
Decreased water consumption was observed in 1000 and 2000 mg/L rats. During the 13-week phase of the study, there was no effect on survival, hematology, or organ weights in rats. Renal tubule regeneration was characterized by hyperplasia of tubular epithelial cells with cytoplasmic basophilia, nuclear crowding, karyomegaly, and occasional mitotic figures. In the rats, these lesions were predominantly found in the proximal convoluted tubules of the cortex. Alterations in urine chemistry parameters were reflective of the renal damage in the high dose groups of rats. Total tungsten concentrations were generally dose proportional in blood and urine. The micronucleus assay was negative in rats. The Comet assay was positive in the liver of rats and negative in the blood and kidney of rats. The kidney appeared to be the only major target organ following exposure of rats to sodium tungstate dihydrate at drinking water cncentrations of 1000 and 2000 mg/L.
Executive summary:

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

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
no data
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well documented, scientifically sound study that was conducted according to EPA OTS 798.2650 (90-Day Oral Toxicity in Rodents)
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium tungstate
Target: Tungsten Trioxide
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Reason / purpose:
reference to same study
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
EPA OTS 798.2650 (90-Day Oral Toxicity in Rodents)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Purchased from Charles River Laboratories, Raleigh, NC
- Age at study initiation: 5 weeks
- Weight at study initiation: about 150 grams when received 199-230 grams at the start of testing)
- Housing: individually housed in polycarbonate cages
- Diet: Harlan Teklad, 8728C Certified Rodent Diet, ad libitum
- Water: ad libitum
- Acclimation period: 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-26 (64-79 degrees F)
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): The light/dark cycle was a 12-hour interval


IN-LIFE DATES: From: no data To: no data
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Sodium tungstate dihydrate was solubilized with deionized (DI) water to produce four dosing solutions of 200, 125, 75, and 10 mg Na2WO4 /mL. This was achieved by placing 224.5, 140.35, 84.20, and 11.23 grams of sodium tungstate dihydrate into 1000 mL volumetric flasks and adding DI water to obtain 1000 mL of solution. Aliquots of test solutions were analyzed for purity and stability by the Aberdeen Test Center and found to be consistent for the purity and stable during the period of studies.

A 90-day stability study on a single suspension of sodium tungstate in DI water was initiated prior to beginning the ALD. This solution was sampled and analyzed weekly for a period of approximately 90 calendar days to ensure that the dosing solutions would remain stable throughout the 90-day study.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Each batch of solutions that was mixed was sampled and analyzed to verify the concentrations prior to use.
Duration of treatment / exposure:
90 days (91 calendar days)
Frequency of treatment:
Tungstate or control solutions were administered daily (7 days per week, total of 90 doses)
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on previous studies;
- Rationale for animal assignment: Randomly distributed using the LABCAT Randomization Program into four treatment groups and one control group.
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- Cage side observations included but were not limited to changes in skin and fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling, as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g., self-mutilation, walking backwards), were recorded. Records indicated time of onset, degree, and duration of all signs. A scoring system for observations explicitly defined by the USACHPPM Toxicity Evaluation Program was used.

- Animals were observed daily for toxic signs.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A careful clinical examination was made for each animal prior to initiation of treatment and once weekly during treatment of animals.

BODY WEIGHT: Yes
- Time schedule for examinations: recorded on days -3, -1, 0 (first day of dosing), 7, and weekly thereafter.


FOOD CONSUMPTION AND COMPOUND INTAKE:
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmic examinations were performed prior to the scheduled start of the 90-day study and within a week of the scheduled necropsies.
- Dose groups that were examined: performed on all control and treated animals


HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the 90 days, before the rats were euthanized
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all animals
- Parameters examined include white blood cell count (WBC), WBC differential (% neutrophils (NEU %N), % lymphocytes (LYM %L), % monocytes (MONO %M), % eosinophils (EOS %E), % basophils (BASO %B), red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelets (PLT), and mean platelet volume (MPV). Blood Coagulation, average prothombin time (AVG PT) and average activated prothombin time (AVG APTT) were analyzed by using MCA 210 Microsample Coagulation Analyzer™ (BioData Corporation, Horsham, Pennsylvania).


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the 90 days, before the rats were euthanized
- Animals fasted: No data
- How many animals: all animals
- The clinical chemistry analytes included: alkaline phosphatase (ALK P), alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), calcium (Ca), cholesterol (CHOL), creatinine kinase (CK), creatinine (CREA), glucose (non-fasting) (GLU), lactate dehydrogenase (LDH), total bilirubin (TBIL), total protein (TP), triglycerides (TRIG), sodium (Na), potassium (K), and chloride (Cl).


URINALYSIS: Yes
- Time schedule for collection of urine: Performed on 8 out of 10 animals from all dose groups (including negative control) within 2 weeks of the final (90-day) necropsies
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Urinalysis was conducted by measuring volume, color, appearance, pH, specific gravity, glucose, bilirubin, urobilinogen, ketone, blood, protein, nitrite, and leukocytes.


NEUROBEHAVIOURAL EXAMINATION: No


OTHER: The brain, heart, liver, kidneys, spleen, adrenals, thymus, epididymides/uterus, and testes/ovaries were removed and weighed for absolute organ weights, organ-to-body weight ratios, and organ-to-brain weight ratios.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes; The tissues harvested for histopathological evaluation included the brain, pituitary, thyroid w/ parathyroid, thymus, lungs, trachea, heart, bone marrow, salivary gland, liver, spleen, kidney, adrenal, pancreas, gonads, uterus, aorta, esophagus, stomach, duodenum, jejunum, ileum, caecum, colon, urinary bladder, lymph node, peripheral nerve, thigh musculature, eye, spinal cord (three levels), and exorbital lachrymal gland.

Following fixation, complete tissues from the control, 125 and 200 mg/kg/day group males and females were trimmed, embedded in paraffin, sectioned at 5 microns, stained with hematoxylin and eosin, and examined via routine light microscopy. Additionally, kidney sections from all animals in the 10 and 75 mg/kg/day dosage groups and all gross lesions were similarly processed. The microscopic sections were of adequate size and quality to allow critical histologic evaluation.
Statistics:
Data from each treatment group were statistically compared to controls using a two-factor ANOVA with sex and dose and sex by dose interaction. When significance was observed, the data were further analyzed using a Dunnett's test to compare the doses to the 0 mg/kg dose. A one-factor ANOVA for each sex was used to see dose differences. Again, a Dunnett's test was used to compare the doses to the control. If a normality test failed, the data were subjected to a log transformation prior to performing ANOVA. If the normality test failed again after the data were transformed, ANOVA on ranks (Kruskal-Wallis test) was performed. Statistical significance was defined at the p< 0.05 level.

Food consumption, body weights, organ-to-brain weight ratios and organ-to-body weight ratios were compared among dosage groups and controls using a one-way analysis of variance (ANOVA) and, if statistical significance was found, Dunnett's post hoc test was used to compare dosage groups to the control group. The parameters were collected with the LABCAT system and statistically analyzed using Sigma-Stat (Sigma-Stat, Jandel Scientific, Corte Madera, CA). Clinical chemistry, hematology, and urinalysis data were entered into Sigma-Stat using a one- way ANOVA and Bonferroni's post hoc test to compare dosage groups to the control group. Where a normality test had failed after the data had been log transformed, an ANOVA on ranks was performed.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
The rats showed no overt toxic or clinical signs during the study


BODY WEIGHT AND WEIGHT GAIN
Significant differences between males and females in overall mean body weights were observed for all days except Day 0. No significant differences between treatment groups (10, 75, 125 and 200 mg/kg/day) in mean body weight were observed for females. However, significant treatment group differences in mean body weight were observed for males on Days 70, 77, 84 and 90. The cntrol group had significantly different mean body weights from the 200 mg/kg group on Days 77, 84 and 90. The 10 mg/kg group had significantly different mean body weights from the 200 mg/kg group on Days 70, 77, 84 and 90. The 75 mg/kg group had significantly different mean body weight from the 200 mg/kg group on Day 77.

The decrease seen was primarily due to significant decreases in liver, heart, testes and epididymis weights of male animals given 200 mg/kg sodium tungstate. This was strongly correlated with a decrease in food consumption in these animals.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The decrease in body weight was strongly correlated with a decrease in food consumption in these animals.

OPHTHALMOSCOPIC EXAMINATION
All observations prior to study initiation were within normal limits. Observations taken within a week of the scheduled necropsies revealed no abnormalities.


HAEMATOLOGY
No significant dose related adverse alterations were observed for hematological parameters for any treatment group.

Significant differences between sexes were observed for WBC, basophils, RBC, MCV, MCH, and RDW. For MCV, MCH and RDW, the females had greater values than the males and for WBC, basophils and RBC the males had greater values than the female. No significant sex by dose interactions or dose group differences were observed.


CLINICAL CHEMISTRY
Since the males and females showed some significant differences in clinical chemistries and there were also significant sex by dose interactions, comparison of dose groups was conducted for males and females separately. For males only, a significant difference between dose groups was observed for creatinine, the 75 mg/kg dose group (0.32±0.02 mg/dL) was significantly less than the 200 mg/kg dose group (0.40±0.02 mg/dL). For females only, significant differences between dose groups were observed for glucose, sodium and chloride. The mean glucose for the 75 mg/kg group (188.8±5.09 mg/dL) was significantly greater than the control group (161.3±6.58 mg/dL). This was also greater than the biological range of 120-186 mg/dL. The mean sodium for the 10 mg/kg group (148±0.27 mmol/L) was significantly greater than the 200 mg/kg group (146±0.38 mmol/L). The mean chloride for the 125 mg/kg group (107±0.47 mmol/L) was significantly greater than the 200 mg/kg group (106±0.37 mmol/L). Sodium and chloride values for all groups, however were within the biological range of 141-148 mmol/l and 101-108 mmol/L, respectively, for Sprague-Dawley rats.

URINALYSIS
Examination of urine samples taken approximately 1 week prior to necropsy revealed no significant changes in volume, specific gravity or pH. No distinct dose-related trends were observed in glucose, bilirubin, ketone, blood, protein, urobilinogen, nitrite, or leukocytes.

ORGAN WEIGHTS
Females given 200 mg/kg had increases in kidney and spleen weight. Histopathology indicated an increase in renal tubular regeneration at this dosage level which may have been responsible for the increase in kidney weight. Most metals were excreted through renal clearance and gastrointestinal excretion. Renal effects are common with heavy metals and this finding was not unexpected.

GROSS PATHOLOGY
Individual animal necropsy observations were reviewed at the time of histologic examination. All macroscopic changes were considered to be spontaneous and/or incidental in nature and unrelated to test article exposure. All tissues evaluated showed no treatment related effects.

HISTOPATHOLOGY: NON-NEOPLASTIC
Individual animal necropsy observations were reviewed at the time of histologic examination. All macroscopic changes were considered to be spontaneous and/or incidental in nature and unrelated to test article exposure.

Following fixation, complete tissues (brain, pituitary, thyroid w/parathyroid, thymus, lungs, trachea, heart, bone marrow, salivary gland, liver, spleen, kidney, adrenal, pancreas, gonads, uterus, aorta, esophagus, stomach, duodenum, jejunum, ileum, caecum, colon, urinary bladder, lymph node, peripheral nerve, thigh musculature, eye, spinal cord (three levels), and exorbital lachrymal gland) from the control, and two high dose groups (125 and 200 mg/kg/day) males and females were trimmed, embedded in paraffin, sectioned at 5 microns, stained with hematoxylin and eosin, and examined via routine light microscopy. Additionally, kidney sections and target tissues (stomach and epididymides) from all animals in the 10 and 75 mg/kg/day dosage groups and all gross lesions were similarly processed. The microscopic sections were of adequate size and quality to allow critical histologic evaluation.

Rats dosed with sodium tungstate showed considerable histopathological changes in the kidneys of male and female rats. Mild to severe regeneration of renal cortical tubules was noted in 1/9 and 10/10 males and 1/10 and 8/10 females in the 125 and 200 mg/kg/day dosage groups, respectively. For clarity, basophilic tubular profiles bearing thickened basement membranes were diagnosed as chronic progressive nephropathy, and in all affected animals, this lesion was minimal and widely scattered throughout the cortex; incidence was similar between control and test article-treated groups, with males more commonly affected than females.

Some histologic findings were noted in the glandular stomach of males and females in all dosage groups. Subacute inflammation consisting primarily of eosinophils admixed with fewer mononuclear cells was observed throughout the submucosa of 2/10, 1/10, 5/9, 4/10 males and 0/10, 1/10, 8/10, 9/10 females in the 10, 75, 125 and 200 mg/kg/day dosage groups, respectively. Goblet cell metaplasia was also observed throughout the mucosa of the glandular stomach in 1/10, 4/10, 8/9, 8/10 males and 0/10, 4/10, 8/10, 10/10 females in the 10, 75, 125 and 200 mg/kg/day dosage groups, respectively.

Histopathologial analysis of epidydimis of male rats dosed with sodium tungstate showed considerable effects at high dose group. Cellular debris within the lumen with and without hypospermia was noted in the epididymides of 3/10 males in the 200 mg/kg/day dosage group. A single male in the 10 mg/kg/day group exhibited a similar lesion; however, the finding in this individual was minimal and unilateral, likely a spontaneous occurrence, and was considered to be unrelated to test article exposure. The lesion was not observed in 75 and 125 mg/kg/day group males.

Although tubular regeneration could be identified within the kidneys of control group animals as well as rats in the 10 and 75 mg/kg/day dosage groups, affected tubules were rare to few and minimally affected; this was considered to be consistent with spontaneous nephropathy syndrome.

Luminal cellular debris was observed in the epididymis of three rats from the 200 mg/kg group. Epididymal changes of this type are commonly encountered as rats reach sexual maturity, and were presumed to represent degenerative cells that were released from the testis. However, rats in the present study should have reached sexual maturity before the time of necropsy. It was interesting to note that two of the rats with the most pronounced epididymal luminal cell debris were found dead or moribund sacrifice rats that died on days 55 and 56, respectively, rather than the scheduled terminal necropsy on study days 90-91. The epididymis of one animal had luminal cell debris that was limited to the tail region, suggesting some transient event that resulted in release of degenerative cells from the testis or epididymis for a defined period of time. There were no identifiable testicular lesions that would explain the presence of degenerated cells in the lumen of the epididymis.
Key result
Dose descriptor:
BMDL10
Effect level:
102 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
LOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
125 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Conclusions:
Sodium tungstate administered orally to male and female Sprague-Dawley rats by gavage for 90 consecutive days induced a number of statistically significant alterations in weights at 200 mg/kg. The administration of sodium tungstate at 125 and 200 mg/kg to male and female Sprague-Dawley rats via oral gavage for 90 consecutive days resulted in pronounced renal changes, specifically mild to severe regeneration of renal cortical tubules. The LOAEL in male and female rats = 125 mg/kg and the NOAEL in male and female rats = 75 mg/kg.
Executive summary:

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

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
BMDL10
102 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The reliability of this study for the substance tested is a K1
System:
urinary
Organ:
kidney

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
650 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
The reliability of this study for the substance tested is a K1, but in application of read-across to a different substance ECHA’s guidance specifies that the score can be a maximum of K2. Due to higher water solubility and greater in vitro bioaccessibility in synthetic alveolar, lysosomal, and interstitial fluids simulating inhalation exposure for the source substance, tungsten blue oxide (TBO) as compared to the target substance (tungsten metal) and lack of toxicity from acute toxicity studies for the target and source substances, toxicity data on the target substance is expected to represent a worse case, so read-across is appropriate between these substances. In addition, read-across is appropriate for this endpoint because the classification and labelling for human health toxicity endpoints is the same for the source and target substances, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, sufficiently similar or more conservative for the target substance. For more details, refer to the attached description of the read-across approach.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Repeated Dose Toxicity - Oral Route:

No oral repeated dose toxicity data of sufficient quality were available for tungsten trioxide; however, data were available for sodium tungstate, which will be used for reading-across. The LOAEL and NOAEL from the 90-day oral toxicity study were deemed to be 125 mg/kg/day and 75 mg/kg/day, respectively, based on histopathological effects reported in the kidneys of the 125 and 200 mg/kg/day dose groups. The BMDL10 derived from this data was calculated to be 102 mg/kg/day. The cutoff range for a category 2 classification under CLP for a 90-day oral toxicity study is between 10 and 100 mg/kg/day. The LOAEL of 125 mg/kg/day identified from the repeat dose oral toxicity study was greater than 100 mg/kg/day. In addition, the benchmark dose (BMDL10) based on the data from the 90-day oral toxicity studies, and using the kidney as the target organ, was calculated to be 102 mg/kg/day. Because the LOAEL from the 90-day study as well as the calculated BMDL10 were greater than the 100 mg/kg/day category 2 cutoff level under CLP, then a classification is not warranted.

Repeated Dose Toxicity - Inhalation Route:

No inhalation repeated dose toxicity data of sufficient quality were available for tungsten trioxide; however, data were available for tungsten oxide, which will be used for reading-across. The cutoff range for a category 2 classification under CLP for a 28-day inhalation toxicity study is between 0.06 and 0.6 mg/L. The NOAEC from the 28-day inhalation toxicity study on tungsten blue oxide was > 0.65 mg/L. Because the NOAEC from the 28-day study was greater than the 0.6 mg/L category 2 cutoff level, classification is not warranted.