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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-11-30 to 2005-05-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2006
Reference Type:
publication
Title:
Subchronic oral toxicity of sodium tungstate in Sprague-Dawley rats
Author:
McCain W.C. et al.
Year:
2015
Bibliographic source:
International Journal of Toxicology Vol 34, Issue 4

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
not specified
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Reference substance name:
Reference substance 001
EC Number:
600-275-2
Cas Number:
10213-10-2
Specific details on test material used for the study:
TEST MATERIAL FORM
- solid: particulate/powder

DETAILS ON TEST MATERIAL
- Name of test material (as cited in study report): sodium tungstate dihydrate
- Molecular formula (if other than submission substance): Na2WO4 2H2O
- Molecular weight (if other than submission substance): 329.85 g/mol
- Analytical purity: 99%
- Lot/batch No.: 12330JO
- Water solubility: 730 g/L at 20 °C

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River laboratories, Raleigh, NC
- Age at study initiation: 5 weeks
- Weight at study initiation: 199 - 230 grams
- Housing: individually housed in polycarbonate cages with one-half inch certified Sani-Chip bedding at the bottom of the cage replaced twice weekly
- Diet (e.g. ad libitum): Harlan Teklad, 8728C Certified Rodent Diet ad libitum
- Water (e.g. ad libitum): drinking quality water ad libitum
- Acclimation period: 1 week in quarantine prior to initiation of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 64-79 degrees Fahrenheit
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
deionized
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.

VEHICLE
- Amount of vehicle (if gavage): 1 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
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
Duration of treatment / exposure:
91 calendar days
Frequency of treatment:
daily (7 days per week, total of 90 doses)
Doses / concentrationsopen allclose all
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
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: selected on the basis of previous studies

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: yes, daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to initiation and once weekly
- A scoring system for observations explicitly defined by the USACHPPM Toxicity Evaluation Program was used.

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to initiation and within a week of scheduled necropsies
- Dose groups that were examined: control and treated animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: following 90-day study period
- Anaesthetic used for blood collection: Yes, with a cocktail of ketamine/xylazine (90 mg/kg ketamine plus 10 mg/kg xylazine intra-muscle via a 22 gauge or smaller needle)
- How many animals: 50 (total)
- Endpoints: Hematological parameters were assayed using Abbott Cell-Dyn 3700 Hematology Analyzer. The parameters 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: following 90-day study period
- How many animals: from all valid samples
- Endpoints: Clinical Chemistry parameters were measured using VetTest 8008 Chemistry Analyzer and VetLyte Na, K, Cl Analyzer. 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 chlorine (Cl).

URINALYSIS: Yes
- Time schedule for collection of urine: within 2 weeks of final (90-day) necropsies
- Endpoints: Urinalysis was conducted by measuring volume, color, appearance, pH, specific gravity, glucose, bilirubin, urobilinogen, ketone, blood, protein, nitrite, and leukocytes.

ORGAN WEIGHTS: Yes
- 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,
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.
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. Body weights, weight gains, food consumption, absolute organ weights, organ-to-body weight ratios, and organ-to-brain weight ratios were also analyzed and compared to controls.
Statistics:
Data from each treatment group were 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 analysed 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. 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 as p less than or equal to 0.05.
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.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
Sodium tungsate when given orally for 90 consecutive days showed no overt toxic or clinical signs during the study
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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 Control 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.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmic examinations were performed on all animals prior to the scheduled start of the 90-day study and within a week of the scheduled necropsies. All observations prior to study initiation were within normal limits. Observations taken within a week of the scheduled necropsies revealed no abnormalities.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
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 biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
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/dl14.
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 findings:
no effects observed
Description (incidence and severity):
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
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. 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 are excreted through renal clearance and gastrointestinal excretion. Renal effects are common with heavy metals and this finding was not unexpected.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
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 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.
Histopathological findings: neoplastic:
not examined
Details on results:
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 Control 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.
OTHER FINDINGS: General - Sodium tungsate when given orally for 90 consecutive days showed no overt toxic or clinical signs during the study. Significant dose related alterations in weights, weight ratios, were observed in animals receiving 200 mg/kg. No significant dose related adverse alterations were observed for hematological and clinical chemistry parameters for any treatment group.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effect observed
Dose descriptor:
LOAEL
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: histopathological changes in the kidneys

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
125 mg/kg bw/day (nominal)
System:
other: kidney
Organ:
kidney
Treatment related:
yes

Applicant's summary and conclusion

Conclusions:
The LOAEL for subchronic oral toxicity in male and female Sprague-Dawley rats is 125 mg/kg and the NOAEL for the subchronic oral toxicity in male and female Sprague-Dawley rats is 75 mg/kg bw/day.
Executive summary:

In a 90-day subchronic toxicity study, Sodium tungstate dihydrate was administered to 10 Sprague-Dawley rats/sex/dose via oral gavage at dose levels of 0, 10, 75, 125, and 200 mg/kg bw/day. All animals were provided drinking quality water and certified laboratory diet ad libitum. Controls were only given the vehicle of deionized water.

The test item showed no overt toxic or clinical signs during the study. Dose related alterations in weights and weight ratios were observed in animals receiving 200 mg/kg bw/day. While females had no significant differences in mean body weights between treatment groups, males had significantly different mean body weights between control groups near the end of the study. Controls, 10 mg/kg bw, and 75 mg/kg bw had significantly different mean body weights than the 200mg/kg bw group in the end of the study. No significant dose related adverse alterations were observed for hematological, clinical chemistry, urinalysis and ophthalmic parameters. 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. There were considerable histopathological changes in the kidneys of male and female rats. Mild to severe regeneration of 18 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. The LOAEL is 125 mg/kg bw/day based on kidney histopathological results.  The NOAEL is 75 mg/kg bw/day.

This subchronic toxicity study in the Sprague-Dawley rat is acceptable and satisfies the guideline requirement for a subchronic oral study (OPPTS 870.3100; OECD 408) in rodents.