Disodium wolframate

Endpoint summary

Description of key information

A repeated dose 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 of sodium tungstate Dihydrate, one in Sprague-Dawley rats and a second one in B6C3F1 mice (10/sex/species/dose). The study design included doses of 0, 125, 250, 500, 1000, or 2000 mg/L. 

There were no early deaths during the 3-month rat study. When compared to the vehicle control group, final mean body weights were lower for the 1,000 and 2,000 mg/L males and 2,000 mg/L females. Water consumption was lower for the 1,000 and 2,000 mg/L males and females. The urine xanthine/creatinine ratios were significantly increased in all male and female exposed groups. Serum insulin concentrations were significantly decreased in the 2,000 mg/L males relative to the vehicle control males. Significantly decreased absolute weights were observed in several organs but were considered secondary to body weights reductions. Exposure-related histological lesions were limited to the kidneys and included increased incidences of renal tubule regeneration in the 1,000 and 2,000 mg/L males and females; the increases in the 2,000 mg/L groups were significant relative to the vehicle control group.

In the mice study, a 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.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

SD

Sex:

male/female

Details on test animals or test system and environmental conditions:

Groups of 10 F1 rats per sex continued on in the study after weaning and were provided drinking water containing the same respective sodium tungstate concentration for 3 months. For all exposure concentrations, except the 2,000 mg/L group, two pups per sex from five randomly selected litters per exposure group were chosen. For the 2,000 mg/L group, a third male pup was selected from two of the four available litters and a third female pup was selected from the other two litters to obtain the complete number of animals needed for the study. After assignments to the 3-month study were complete, five pups per sex from the remaining vehicle control pups were randomly selected as the end-of-study sentinel animals. On the day the last litter reached PND 21, dams were removed, and the pups were weaned. Weaning marked the beginning of the 3-month study.
After weaning, F1 rats were housed five per cage. Feed and dosed water were available ad libitum. Water consumption was measured weekly for 3 months. Cages were changed weekly though PND 4, then changed twice weekly. Racks were changed and rotated at least every 2 weeks.

Route of administration:

oral: drinking water

Details on route of administration:

deionized 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

Remarks:

Approximately 11.8 mg/kg/day for males and 14.0 mg/kg/day for females.

Dose / conc.:

250 mg/L drinking water

Remarks:

Approximately 24.3 mg/kg/day for males and 26.1 mg/kg/day for females.

Dose / conc.:

500 mg/L drinking water

Remarks:

Approximately 48.9 mg/kg/day for males and 54.4 mg/kg/day for females.

Dose / conc.:

1 000 mg/L drinking water

Remarks:

Approximately 91.8 mg/kg/day for males and 101.4 mg/kg/day for females.

Dose / conc.:

2 000 mg/L drinking water

Remarks:

Approximately 157.2 mg/kg/day for males and 160.5 mg/kg/day for females.

No. of animals per sex per dose:

Each group per sex per species contains five animals

Control animals:

yes, concurrent vehicle

Details on study design:

On the day the last litter (exposed from GD 6 to GD21) reached PND 20, pups were randomly assigned to the 3-month study. For all exposure concentrations, except the 2,000 mg/L group, two pups per sex from five randomly selected litters per exposure group were chosen. For the 2,000 mg/L group, a third male pup was selected from two of the four available litters and a third female pup was selected from the other two litters to obtain the complete number of animals needed for the study. After assignments to the 3-month study were complete, five pups per sex from the remaining vehicle control pups were randomly selected as the end-of-study sentinel animals. On the day the last litter reached PND 21, dams were removed, and the pups were weaned. Weaning marked the beginning of the 3-month study.
After weaning, F1 rats were housed five per cage. Feed and dosed water were available ad libitum. Water consumption was measured weekly for 3 months. Cages were changed weekly though PND 4, then changed twice weekly. Racks were changed and rotated at least every 2 weeks.

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 samples were taken from rats at study termination for micronuclei determinations.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical observations related to exposure, and all exposed animals were similar in overt behavior and general appearance to the vehicle control animals

Mortality:

no mortality observed

Description (incidence):

There were no early deaths during the 3-month study

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Initial mean body weights were 9% and 16% below those of the vehicle control group for the 1,000 and 2,000 mg/L males, respectively; and 14%, 11%, and 13% below those of the vehicle control group for the 500, 1,000, and 2,000 mg/L females, respectively. Final mean body weights were lower for the 1,000 and 2,000 mg/L males and females, with the 2,000 mg/L males weighing approximately 29% less than the vehicle control group and the 2,000 mg/L females weighing approximately 18% less than the vehicle control group.

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):

Water consumption was lower for the 1,000 and 2,000 mg/L males and females, with overall reductions of 27% and 42% for males and females, respectively, in the 2,000 mg/L groups compared to the respective vehicle control groups.

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In female rats, there was a mild (<10%) significant decrease in the erythron characterized by a significant decrease in the hemoglobin concentration in the 2,000 mg/L group and a significant negative trend in the hematocrit concentration, hemoglobin concentration, and erythrocyte count with increasing exposure. Although there were no significant pairwise changes observed in the male erythron, there were significant negative trends in hematocrit concentration, hemoglobin concentration, and erythrocyte count with increasing exposure concentration. The reticulocyte count was unchanged in both males and females. These mild erythron changes were most likely due to the stress of exposure,
which is supported by the lower mean body weights
observed in the 2,000 mg/L groups.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In male rats, blood urea nitrogen (BUN) was significantly increased, and the total protein, globulin concentrations, and insulin concentrations were significantly decreased in the 2,000 mg/L group. The BUN was likely increased due to the lower water consumption values in that exposure group. The toxicological relevance of the observed decreases in the total protein and globulins is uncertain; these changes could be a secondary effect of exposure.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The urine xanthine/creatinine ratios were significantly increased in all male and female exposed groups relative to the vehicle control groups

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute kidney weights were reduced in males in all exposed groups, relative to the vehicle control group, with a significant decrease observed in the 2,000 mg/L group (approximately 21%). Relative kidney weights were higher in 1,000 mg/L females and significantly increased in the 2,000 mg/L males and females, relative to the vehicle control group. Although the kidney was a target tissue, it is unlikely that the lesions observed were responsible for the differences in kidney weights; it is more likely that these organ weight differences are an effect of body weight differences.
When compared to vehicle control groups, significant differences were also observed in other organ weights, including decreased absolute heart and lung weights in males and females; decreased absolute liver weights in males and increased relative liver weights in females; decreased absolute thymus weights in males and increased relative testis weights. These changes were considered secondary to body weight reductions.
Rats administered 2,000 mg/L exhibited significantly decreased left cauda epididymis (14%) and epididymis (13%) weights, and lower testis weights (8%) compared to the vehicle control group.
Although these were significant (cauda and epididymis) and/or displayed a significant negative trend with increasing exposure concentration (right testis), rats in the 2,000 mg/L group displayed mean body weights that were 28% lower than the vehicle control group. There were no changes in reproductive parameters or alterations in contralateral testis and epididymis or in histopathology. Given the magnitude of the body weight effect and the absence of changes in other endpoints, the lower reproductive organ weights are likely secondary to effects on body weight.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No exposure-related gross lesions were recorded

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Exposure-related histological lesions were found in the kidneys. Renal tubule regeneration was increased in the male and female 1,000 and 2,000 mg/L groups; the increases in the 2,000 mg/L groups were significant relative to the vehicle control groups. The lesion was characterized by hyperplasia of proximal convoluted tubular epithelial cells that manifested as cytoplasmic basophilia, nuclear crowding, and occasional mitotic figures. Renal tubule regeneration occurs as a response to previous degeneration or necrosis and is one of the most common exposure-related lesions in the kidney.90
Degeneration and necrosis were not present in this study, perhaps due to the fact that by the time of necropsy, the response of the kidney had progressed from degeneration to regeneration. Renal tubule regeneration differed from chronic progressive nephropathy (CPN) by the lack of thickened basement membranes, associated inflammatory cells, proteinaceous casts, and cytoplasmic pigment—all features typically seen with CPN. The incidences and severities of CPN were not increased in exposed groups of animals.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Neoplasms were not identified in male or female rats

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Although the weights of the left epididymis and the left cauda were significantly decreased in the 2,000 mg/L males, there were no corresponding changes in sperm parameters, including number of sperm/mg cauda epididymis, total number of sperm/cauda, sperm motility, number of homogenization-resistant spermatids/mg testis, or total number of spermatids.

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

In both males and females, the total tungsten concentration in blood increased proportionally to the exposure concentration with no observed sex difference. The blood tungsten concentration in vehicle control animals was below the limit of detection (LOD; 0.0016 μg/g) of the assay. The urine tungsten concentration is presented as both μg/g of urine and after correcting for urinary creatinine concentrations (μg/mg creatinine). Low concentrations of tungsten were detected in urine from vehicle control male and female groups. The concentrations of creatinine-corrected tungsten in urine increased proportionally to the exposure concentration in both males and females and were significantly increased in all exposed groups compared to the corresponding vehicle control groups. As with blood, there was no observed sex difference in urinary tungsten concentrations.
The micronucleus assay was negative in rats. The Comet assay was positive in the liver of rats but negative in the blood and kidney.

Conclusions:

There were no early deaths during the 3-month study. When compared to the vehicle control group, final mean body weights were lower for the 1,000 and 2,000 mg/L males and 2,000 mg/L females. Water consumption was lower for the 1,000 and 2,000 mg/L males and females. The urine xanthine/creatinine ratios were significantly increased in all male and female exposed groups. Serum insulin concentrations were significantly decreased in the 2,000 mg/L males relative to the vehicle control males. Significantly decreased absolute weights were observed in several organs but were considered secondary to body weights reductions. Exposure-related histological lesions were limited to the kidneys and included increased incidences of renal tubule regeneration in the 1,000 and 2,000 mg/L males and females; the increases in the 2,000 mg/L groups were significant relative to the vehicle control group.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female B6C3F1/N mice were 4 to 5 weeks old upon receipt and were quarantined for 11 days before study start. Mice were randomly assigned to one of six exposure groups (n = 10 mice/sex/group).
Mice were housed individually (males) or five per cage (females). Feed and dosed water were available ad libitum. Water consumption was measured weekly for 3 months. Cages were changed at least once weekly (males) or twice weekly (females) and rotated every 2 weeks. Racks were changed and rotated every 2 weeks.

Route of administration:

oral: drinking water

Details on route of administration:

deionized drinking water

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulations were stable for 42 days at room temperature. Preadministration and postadministration (animal room) analyses of dose formulations were conducted monthly throughout the 3-month studies.ll preadministration formulations in the 3-month mouse studies were within 10% of the target concentration. In the 3-month mouse study, four postadministration samples were more than 10% below the target concentration, with the largest difference being 12.8% below the target. Three postadministration samples collected from carboys or bottles for the 125, 500, and 2,000 mg/L dose formulations in the 3-month rat study were 10.8% to 12.3% below the corresponding target concentration.

Duration of treatment / exposure:

90 days (3-months)

Frequency of treatment:

Daily

Dose / conc.:

0 mg/L drinking water

Dose / conc.:

125 mg/L drinking water

Remarks:

Average daily sodium tungstate dose of approximately 14 mg/kg/day for males and females.

Dose / conc.:

250 mg/L drinking water

Remarks:

Average daily sodium tungstate dose of approximately 27 mg/kg/day for males and 29 mg/kg/day for females.

Dose / conc.:

500 mg/L drinking water

Remarks:

Average daily sodium tungstate dose of approximately 57 mg/kg/day for males and 58 mg/kg/day for females.

Dose / conc.:

1 000 mg/L drinking water

Remarks:

Average daily sodium tungstate dose of approximately 108 mg/kg/day for males and 113 mg/kg/day for females.

Dose / conc.:

2 000 mg/L drinking water

Remarks:

Average daily sodium tungstate dose of approximately 212 mg/kg/day for males and 202 mg/kg/day females.

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):

There were no early deaths or exposure-related clinical observations in male or female B6C3F1/N mice exposed to sodium tungstate for 3 months

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Over the course of the study, group mean body weights were below 90% of the vehicle control group mean for the 250, 1,000, and 2,000 mg/L females and the 2,000 mg/L males. At study termination, the mean body weights of all exposed groups of males and females were within 10% of the vehicle control groups.

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):

Weekly mean water consumption was reduced slightly in the 1,000 mg/L male group (11%), the 2,000 mg/L males (16%), and the 2,000 mg/L females (11%), relative to the respective vehicle control groups

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In male mice, the white blood cell count was significantly decreased in the 1,000 and 2,000 mg/L groups relative to the vehicle control group (Table 36). These decreases were driven by a significant decrease in the lymphocyte count in the 1,000 and 2,000 mg/L groups and a significant decrease in the monocyte count in the 500 mg/L and higher groups. Additionally, the eosinophil counts were significantly decreased in all sodium tungstate-exposed male groups. These leukocyte changes are consistent with a stress leukogram (i.e., effects of chronic increase in endogenous corticosterone.

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:

effects observed, treatment-related

Description (incidence and severity):

A higher group mean relative testis weight was observed in the 2,000 mg/L male group, relative to the vehicle control group, and was likely due to the lower mean body weights in that group. There were no histological lesions in the testes related to sodium tungstate exposure. In female mice, there were several sporadic increases in group mean organ weights, but they lacked an exposure concentration response or other supporting evidence that they represented anything but biological variation. Relative kidney weights were significantly increased in the 1,000 and 2,000 mg/L females and 2000 mg/L males but were most likely due to reduced mean body weights in those groups compared to the vehicle control group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No exposure-related gross lesions were recorded

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The only histological lesion associated with exposure was in the kidney. The incidences of renal tubule regeneration were higher in the 1,000 and 2,000 mg/L male and female groups compared to the respective vehicle control groups; the increases in the male groups were significant. The lesion consisted of hyperplastic tubules, predominantly in the deep cortical to medullary region, lined by epithelial cells with increased cytoplasmic basophilia, nuclear crowding, prominent nucleoli, marginated chromatin, and karyomegaly. There were occasional mitotic figures.

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):

In male mice, no significant differences were observed between exposed groups and the vehicle control group in left testis weight, left epididymal weight, left cauda weight, or any of the sperm parameters, including number of sperm/mg cauda epididymis, total number sperm/cauda, sperm motility, number of homogenization-resistant spermatids/mg testis, and total number of spermatids. The testes and epididymides were evaluated to a no-effect level, and no histological findings associated with sodium tungstate exposure were present at 3 months. Under the conditions of this 3-month study, Sodium tungstate administration via drinking water did not exhibit the potential to be a reproductive toxicant in B6C3F1/N 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

Blood was collected from up to 10 animals per group on the morning of day 91. Total blood tungsten concentrations were determined using a validated analytical method. Tungsten was not detected in males in the vehicle control group above the LOD of the assay (0.0016 μg/g); however, low concentrations of tungsten were detected in females in the vehicle control group. In both males and females, the blood tungsten concentrations increased proportionally with the exposure concentration; there was no observed sex difference. In females, the tungsten concentrations in exposed groups were significantly higher than in the corresponding vehicle control group.

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.

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.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting 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)

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

EPA OTS 798.2650 (90-Day Oral Toxicity in Rodents)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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.

Reference 4

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

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)

Reason / purpose for cross-reference:

assessment report

Qualifier:

according to guideline

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 or test system 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:

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:

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

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 study available

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

On a rabbit 7-day maximum tolerable dose (MTD) study death was observed at 300 mg/kg/day and progressive weight loss at 200 mg/kg/day were reported suggesting that the oral MTD oral dose of sodium tungstate in the non-pregnant rabbit was less than 200 mg/kg/day.  Doses of 100 or 50 mg/kg/day were generally well tolerated with only small fluctuations in body weight. A high dose level between 100 and 200 mg/kg/day is concluded to be appropriate for the subsequent rabbit studies.

Justification for classification or non-classification

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.