Registration Dossier

Administrative data

Description of key information

Repeated Dose Toxicity - Oral Route:

No oral repeated dose toxicity data of sufficient quality are available for the RM of W2C + WC (target substance). However, repeated oral dose toxicity data are available for sodium tungstate (source substance), which will be used for reading across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

The read across study on sodium tungstate was sponsored conducted the United States Army Center for Health Promotion and Preventive Medicine and published byMcCain 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 epididymis included intraluminal hypospermia with cell debris in the 200 mg/kg bw/day dosed male rats. Histopathological changes were observed in the glandular stomach including inflammation and metaplasia in the high-dose groups (125 or 200 mg/kg bw/day) of both sexes of rats. Based on histopathology effects seen in the kidneys, the lowest observable adverse effect level was 125 mg/kg bw/d and the no observable adverse effect level was 75 mg/kg bw/d in both sexes of rats for oral subchronic toxicity. The USEPA’s Benchmark Dose Software (BMDS, Version 1.4.1) was used to model the data to derive a BMDL10. The lowest (most precautionary) BMDL10 from the renal toxicity endpoint in the 90-day oral toxicity study was 102 mg/kg bw/d.

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

Repeated Dose Toxicity - Inhalation Route:

No inhalation repeated dose toxicity data of sufficient quality are available for the RM of W2C + WC (target substance). However, inhalation repeated dose toxicity data are available for tungsten carbide and tungsten oxide (source substances), which will be used for reading across. A 90-day repeat dose inhalation toxicity study was identified for WC in which one dose of WC (15 mg/m3) was administered to rats and mice, whole body inhalation exposure, 5 days/week for 13 weeks. The LOEL was deemed to be 15 mg/m3in rats based on mild histopathological alterations in the lungs (focal reactions around the end airways) and chronic rhinitis, and in mice based on chronic rhinitis. However, because only one dose was evaluated, this dose did not result in any effects on which a DNEL would be derived or classification would be based, and using this dose would result in an inaccurate DNEL and classification, the 28-day inhalation toxicity study on TBO will be used for reading across. In this 28-day inhalation toxicity study conducted according to OECD 412, 5 rats/sex/dose were given TBO nose-only for 6 hours per day, 7 days/week, for 28 days (with a 14-day recovery period) at doses of 0 (control), 0.08, 0.325, and 0.65 mg TBO/L air. The NOAEL was deemed to be > 0.65 mg/L air (650 mg/m3), as no significant effects were reported.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well documented, scientifically sound study that was conducted according to EPA OTS 798.2650 (90-Day Oral Toxicity in Rodents)
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium tungstate
Target: RM W2C + WC
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Reason / purpose:
assessment report
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
EPA OTS 798.2650 (90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Sodium tungstate dihydrate (Tungstic acid sodium salt dihydrate, Na2WO42H2O, CAS # 10213-10-2, Batch # 12330JO) 99% pure
Species:
rat
Strain:
other: SD
Details on species / strain selection:
5-week-old SD rats were purchased from Charles River laboratories, Raleigh, North Carolina
Sex:
male/female
Details on test animals and environmental conditions:
Animals were held for 1 week in quarantine prior to initiation of treatments. At the start of testing, rats weighed between 199 and 230 g. Test animals were identified by individual cage cards and microchip implants and were individually housed in
polycarbonate cages. Bedding was placed in the bottom of each cage and replaced twice weekly. Drinking quality water and a certified laboratory diet were available ad libitum. Animal rooms were maintained at 64 F to 79 F, with relative humidity of 30% to 70% and a 12-hour light–dark cycle.
Route of administration:
oral: drinking water
Details on route of administration:
Sodium tungstate dihydrate was solubilized with DI water to produce 4 dosing solutions
Vehicle:
water
Details on oral exposure:
The dose levels were selected on the basis of our previous studies where the highest dose used was 200 mg/kg/ d in a subchronic toxicity study. Sodium tungstate dihydrate was solubilized with DI water to produce 4 dosing solutions of
200, 125, 75, and 10 mg Na2WO4/mL.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Tungstate concentrations of the dosing solutions were verified by the Aberdeen Test Center and found to be consistent for purity and stability during the study period
Duration of treatment / exposure:
A 90-day oral toxicity study
Frequency of treatment:
Test chemical solutions were administered daily (7 days per week) for 90 days. A 16 GA 2-in stainless steel gavage needle
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Following 1-week quarantine/acclimatization period, 50 male and 50 female SD rats were randomly distributed
Control animals:
yes, concurrent vehicle
Details on study design:
Sodium tungstate dihydrate was solubilized with DI water to produce 4 dosing solutions of 200, 125, 75, and 10 mg Na2WO4/mL.
Observations and examinations performed and frequency:
A clinical examination was made for each animal prior to initiation of treatment and once weekly during treatment. Observations included but were not limited to changes in skin and fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (eg, lacrimation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture, and response to handling as well as the presence of clonic or tonic movements, stereotypes (eg, excessive grooming, repetitive circling), or bizarre behavior (eg, selfmutilation, walking backward) were recorded according to testing guidelines. Body and feeder weights were recorded on days .3, .1, 0 (first day of dosing), 7, and weekly thereafter. Doses were adjusted weekly to reflect the change in individual body weights. Animals were observed daily for any toxic signs.
Sacrifice and pathology:
Blood was collected. Each rat was then submitted for complete necropsy. The brain, heart, liver, kidneys, spleen, adrenals, thymus, epididymis/uterus, and testes/ovaries were removed and weighed for absolute organ weights. The tissues harvested for histopathological evaluation included the brain, pituitary, thyroid with parathyroid gland, thymus, lungs, trachea, heart, bone marrow, salivary gland, liver, spleen, kidney, adrenal gland, pancreas, testis, ovaries, uterus, aorta, esophagus, stomach, duodenum, jejunum, ileum, caecum, colon, urinary bladder, salivary lymph node peripheral nerve (siatic), thigh musculature (vastus lateralis), eye, spinal cord (3 levels), and exorbital lachrymal gland.

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

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

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

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

No oral repeated dose toxicity data of sufficient quality are available for the RM of W2C + WC (target substance). However, oral repeated dose toxicity data are available for sodium tungstate (source substance), which will be used for reading across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium tungstate
Target: RM W2C + WC
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Details on species / strain selection:
B6C3F1/N
Sex:
male/female
Details on test animals and environmental conditions:
After a 10- to 14-day quarantine period, animals are assigned at random to treatment groups including:
- Five treatment groups, each administered a different concentration of the test substance
- One control group

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

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

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

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

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

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

No oral repeated dose toxicity data of sufficient quality are available for the RM of W2C + WC (target substance). However, oral repeated dose toxicity data are available for sodium tungstate (source substance), which will be used for reading across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Sodium tungstate
Target: RM W2C + WC
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Reason / purpose:
reference to same study
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
After a 10- to 14-day quarantine period, animals are assigned at random to treatment groups including:
- Five treatment groups, each administered a different concentration of the test substance
- One control group

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

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

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

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

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

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

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

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

No oral repeated dose toxicity data of sufficient quality are available for the RM of W2C + WC (target substance). However, oral repeated dose toxicity data are available for sodium tungstate (source substance), which will be used for reading across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

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

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1983-05-05 to 1986-02-01
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Similar to OECD Guideline 413 with the following deviations: (1) Only one concentration was tested rather than 3: (2) 8 animals/sex/dose were evaluated rather than 10/sex/dose; (3) ophthalmological and clinical chemistry evaluations were not conducted; (4) histopathology did not include bone marrow evaluation.
Justification for type of information:
1. HYPOTHESIS FOR THE CATEGORY APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Tungsten Carbide
Target: RM W2C + WC
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
(1) Only one concentration was tested rather than 3: (2) 8 animals/sex/dose were evaluated rather than 10(3) ophthalmological and clinical chemistry evaluations were not conducted; (4) histopathology did not include bone marrow evaluation.
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Simonsen Laboratories (Gilroy, CA)
- Age at study initiation: 9-11 weeks
- Housing: individually in stainless steel, wire-mesh cages
- Diet: ad libitum (except during exposure periods)
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1983-05-09 to 1983-06-09 To: 1983-08-02 to 1983-09-02
Route of administration:
inhalation: dust
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: MMAD = 4.2 um with a GSD = 1.86
Details on inhalation exposure:
- The tungsten particles were aerosolized using model 9310 generators. The automatic feed systems of the generators were not employed because the physical consistency of these powders rendered the feed mechanisms ineffective. Instead, each powder was mixed with bronze beads from the respective generator by vigorously shaking in a plastic container. During the mixing process, the bronze beads were coated with dust particles. To disperse the particles, dry, filtered air was introduced through the microporous stainless steel support screen at the bottom of the bed. he air caused the beads to Collide with one another, striping the particles from the beads and carrying the resultant aerosol to the outlet of the generator. The delivery line from each generator leading to the air intake line of the exposure chambers was equipped with a 60 mCi 85Kr neutralizing source to bring the electrical charge of the particles to Boltzman equilibrium.
- Separate generators were used to aerosolize the WC dust in each chamber.
- Exposures were carried out in 5.0 m Hinners type chambers constructed of stainless steel and Lucite. Airflow through the chambers was 11 chamber volumes per hour. Exhaust air from the chambers was passed through electrostatic precipitators, a prefilter and a HEPA filter before being discharged. Temperature in each chamber was monitored continuously by computer and the average temperature during each 0 .5 hr interval recorded. The average temperature during the exposure of these animals was 20 .5 degree C . The mean average daily temperature ranged from 19 .2 to 22 .7 degree C and the minimum and maximum 0 .5 hr averages were 18 .6 and 23 .9 degree C, respectively .
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On-line monitoring of the aerosol mass in the exposure chambers was accomplished using RAM-1 mass monitors and strip chart recorders. During each daily exposure period, at least one but more frequently 2, filter samples were drawn from each exposure chamber. The total dust concentrationsin the WC chambers were determined gravimetrically. The concentration of WC was determined by difference .
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Remarks:
Doses / Concentrations:
15 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
14.97 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
8 mice/sex were designated for observation of body weight changes, organ weights, hematology, and pathology. Another 10 male mice were designated for cytogenetic and sperm abnormality studies.
Control animals:
yes, sham-exposed
Details on study design:
Post-exposure period: 6 days
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily prior to exposure, when the food troughs were removed and clean catch pans were provided, and again following the exposure period when the food troughs were replaced.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On the days that the animals were weighed.

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed in the morning of its initial exposure and then weekly on Mondays and Wednesdays and on the morning following their final exposure and on the day of the endpoint assessment.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At sacrifice
- Anaesthetic used for blood collection: Yes
- Animals fasted: No data
- How many animals: No data
- Parameters that were examined: white and red blood cell counts, hemoglobin, hematocrits, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration.

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, 8 animals/sex/dose
HISTOPATHOLOGY: Yes, Brain, pituitary, adrenals, trachea, esophagus, thyroid, parathyroids, thymus, heart, pancreas, spleen, kidneys, liver, preputial glands, gall bladder, larynx, peribronchial lymph node, lungs, uterus, testis, prostate, epididymis, urinary bladder, stomach, duodenum, jejunum, ileum cecum, colon, salivary gland, skin, mammary gland, mesenteric lymph node, nasal turbinate, sternum, rib junction, skeletal muscle, peripheral nerve, eyes, diaphragm, and penis
Other examinations:
Cytogenetic and sperm abnormality studies.
Statistics:
Growth of male and female exposure groups were analyzed separately. Variation in body weights was examined by analysis of covariance with repeated measures, in which the exposure groups represented the treatment factors, weekly weights were the repeated measures, and initial weights were considered the covariants. Differences in growth rates among the exposure groups were investigated by finding least squares linear regression equations that expressed the mean weekly adjusted weight for each group as a function of exposure time. To statistically assess the differences of means of a single variable between any 2 groups the Student's-t test was employed. However, one way analysis of variance (ANOVA) was used to compare the means of single variables across exposure groups. When ANOVA indicated a significant difference among group means, Duncan's multiple range method of multiple comparisons was used to investigate the source of the differences (p = 0 .05). In addition to ANOVA, quasi-static compliance data and flow-volume data were each analyzed as sets of variables. These sets were compared among exposure groups by a multi-variate analysis of variance (MANOVA). To investigate differences among exposure groups based on histopathologic data, the values were non-parametrically ranked and were then analyzed by the Kruskal-Wallis non-parametric test. When a significant difference was indicated among the groups, non-parametric multiple comparisons were performed according to the Mann-Whitney Simultaneous Test Procedure to identify the source of the differences.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
not specified
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:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
The incidence of chronic runny nose (rhinitis) in female mice was 0/8 controls and 4/8 of the WC group. Other changes in the tissues of the female exposure groups appeared to be incidental. No significant effects on hematological parameters were reported.

No effects were seen in males.

Bone marrow was not evaluated as part of the general histopathology evaluation, but subsets of rats were exposed and bone marrow was removed from the femurs and slides were prepared for a genetic toxicity evaluation. However, the slides prepared were not reviewed.

Dose descriptor:
LOEL
Effect level:
15 mg/m³ air (nominal)
Sex:
female
Basis for effect level:
other: Chronic rhinitis in females
Dose descriptor:
NOAEL
Effect level:
>= 15 mg/m³ air (nominal)
Sex:
male
Basis for effect level:
other: no apparent effects
Critical effects observed:
not specified
Conclusions:
The LOAEL for females was 15 mg/m3 based on chronic rhinitis. No effects were seen in males exposed at this dose level therefore the NOAEL for males was determined to be >=15 mg/m3.
Executive summary:

No inhalation repeated dose toxicity data of sufficient quality are available for the RM of W2C + WC (target substance). However, inhalation repeated dose toxicity data are available for sodium tungstate (source substance), which will be used for reading across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Similar to OECD Guideline 413 with the following deviations: (1) Only one concentration tested rather than 3: (2) 8 animals/sex/dose were evaluated rather than 10/sex/dose; (3) ophthalmological and clinical chemistry evaluations were not conducted; (4) histopathology did not include bone marrow evaluation.
Justification for type of information:
1. HYPOTHESIS FOR THE CATEGORY APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Tungsten Carbide
Target: RM W2C + WC
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
See rationale for reliability above
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Simonsen Laboratories (Gilroy, CA)
- Age at study initiation: 9-11 weeks
- Housing: individually in stainless steel, wire-mesh cages
- Diet: ad libitum (except during exposure periods)
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: dust
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: MMAD = 4.2 um (SW = 0.2 um) with a GSD = 1.86 (SE = 0.10)
Details on inhalation exposure:
- The tungsten particles were aerosolized using model 9310 generators. The automatic feed systems of the generators were not employed because the physical consistency of these powders rendered the feed mechanisms ineffective. Instead, each powder was mixed with bronze beads from the respective generator by vigorously shaking in a plastic container. During the mixing process, the bronze beads were coated with dust particles. To disperse the particles, dry, filtered air was introduced through the microporous stainless steel support screen at the bottom of the bed. he air caused the beads to Collide with one another, striping the particles from the beads and carrying the resultant aerosol to the outlet of the generator. The delivery line from each generator leading to the air intake line of the exposure chambers was equipped with a 60 mCi 85Kr neutralizing source to bring the electrical charge of the particles to Boltzman equilibrium.
- Separate generators were used to aerosolize the WC dust in each chamber.
- Exposures were carried out in 5.0 m Hinners type chambers constructed of stainless steel and Lucite. Airflow through the chambers was 11 chamber volumes per hour. Exhaust air from the chambers was passed through electrostatic precipitators, a prefilter and a HEPA filter before being discharged. Temperature in each chamber was monitored continuously by computer and the average temperature during each 0 .5 hr interval recorded. The average temperature during the exposure of these animals was 20 .5 degree C . The mean average daily temperature ranged from 19 .2 to 22 .7 degree C and the minimum and maximum 0 .5 hr averages were 18 .6 and 23 .9 degree C, respectively .
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On-line monitoring of the aerosol mass in the exposure chambers was accomplished using RAM-1 mass monitors and strip chart recorders. During each daily exposure period, at least one but more frequently 2, filter samples were drawn from each exposure chamber. The total dust concentrations in the WC chambers were determined gravimetrically.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Remarks:
Doses / Concentrations:
15 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
14.97 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
24 male rats were designated for respiratory physiology studies. After pulmonary testing these animals were sacrificed and the left lung was processed for pathological examination while the right lung was submitted for biochemical analysis.

8 rats/sex were designated for observation of body weight changes, organ weights, hematology and pathology.

10 male rats were designated for cytogenetic and sperm abnormality studies (these endpoints were not reported).
Control animals:
yes
Details on study design:
Post-exposure period: 6 days
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily prior to exposure, when the food troughs were removed and clean catch pans were provided, and again following the exposure period when the food troughs were replaced.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On the days that the animals were weighed.

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed in the morning of its initial exposure and then weekly on Mondays and Wednesdays and then on the morning following their final exposure and on the day of the endpoint assessment.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At sacrifice
- Anaesthetic used for blood collection: Yes
- Animals fasted: No data
- How many animals: No data
- Parameters that were examined: white and red blood cell counts, hemoglobin, hematocrits, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: Pulmonary function tests
- Number of animals: 8 male rats/dose
- Parameters: tidal breathing, heart rate, lung volumes, pressure volume characteristics, diffusion capacity, multi-breath nitrogen washout, maximumexpiratory flow volume, vital capacity. Following pulmonary function tests, animals were sacrificed and the right lung was evaluated for biochemical parameters and the left lung was evaluated pathologically.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, 8 animals/sex/dose
HISTOPATHOLOGY: Yes, Brain, pituitary, adrenals, trachea, esophagus, thyroid, parathyroids, thymus, heart, pancreas, spleen, kidneys, liver, clitorial glands, larynx, peribronchial lymph node, lungs, uterus, ovaries, testis, prostate, epididymis, urinary bladder, stomach, duodenum, jejunum, ileum cecum, colon, salivary gland, skin, mammary gland, mesenteric lymph node, nasal turbinate, sternum, rib junction, diaphragm, and penis.
Other examinations:
Animals undergoing pulmonary testing were sacrificed and the left lung was processed for pathological examination while the right lung was submitted for biochemical analysis (weight, water content, protein, DNA, elastin, collagen).

10 male rats were designated for cytogenetic and sperm abnormality studies (these endpoints have not been evaluated).
Statistics:
Growth of male and female exposure groups were analyzed separately. Variation in body weights was examined by analysis of covariance with repeated measures, in which the exposure groups represented the treatment factors, weekly weights were the repeated measures, and initial weights were considered the covariants. Differences in growth rates among the exposure groups were investigated by finding least squares linear regression equations that expressed the mean weekly adjusted weight for each group as a function of exposure time. To statistically assess the differences of means of a single variable between any 2 groups the Student's-t test was employed. However, one way analysis of variance (ANOVA) was used to compare the means of single variables across exposure groups. When ANOVA indicated a significant difference among group means, Duncan's multiple range method of multiple comparisons was used to investigate the source of the differences (p = 0 .05). In addition to ANOVA, quasi-static compliance data and flow-volume data were each analyzed as sets of variables. These sets were compared among exposure groups by a multi-variate analysis of variance (MANOVA). To investigate differences among exposure groups based on histopathologic data, the values were non-parametrically ranked and were then analyzed by the Kruska-Wallis non-parametric test. When a significant difference was indicated among the groups, non-parametric multiple comparisons were performed according to the Mann-Whitney Simultaneous Test Procedure to identify the source of the differences.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
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:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
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:
No effects on the pulmonary function tests were reported in any exposure group. No significant effects on hematological parameters were reported. During the 13 week exposure period the control and all of the dust exposed rats gained weight at the same rate. There were no significant changes in fresh organ weights among any of the male Fischer-344 rat exposure groups. The mean lung weight of the WC subgroup was identical to that of the controls. Also in females it appeared that WC alone had no effect on this index of pulmonary toxicity. All the lung composition variables measured were similar between control and WC treated animals.

Lesions observed in rats exposed to WC consisted of focal reactions around the end airways of the lung. These were characterized by alveolar wall thickening with an increase in the number (hyperplasia) of type II cells. Pigmented and/or foamy macrophages accompanied this end airway change. Although chronic rhinitis was not detected in any of the female rat exposure groups, the incidence in male rats appeared to be related to exposure with 0/8 of the controls and 6/7 of the WC group showing lesions. This change was characterized by submucosal infiltration of mononuclear cells in the nasal cavity. Changes in the lungs of the multiple endpoint rat exposure groups were similar to those of the rats designated for pathology evaluation. Changes observed in the non-respiratory tissues of the rats in the pathology evaluation appeared to be spontaneous lesions and/or incidental findings.
Dose descriptor:
LOEL
Effect level:
15 mg/m³ air (nominal)
Sex:
male
Basis for effect level:
other: Mild histopathological alterations in the lungs (focal reactions around the end airways) and chronic rhinitis.
Dose descriptor:
NOAEL
Effect level:
>= 15 mg/m³ air (nominal)
Sex:
female
Basis for effect level:
other: No apparent effects
Critical effects observed:
not specified

Bone marrow was not evaluated as part of the general histopathology evaluation, but subsets of rats were exposed and bone marrow was removed from the femurs and slides were prepared for a genetic toxicity evaluation. However, the slides prepared were not reviewed.

Conclusions:
The LOAEL for male rats was determined to be 15 mg/m3 based on mild histopathological alterations in the lung (focal reactions at the end airways) and chronic rhinitis, while an NOAEL for females was determined to be >=15 mg/m3.
Executive summary:

No inhalation repeated dose toxicity data of sufficient quality are available for the RM of W2C + WC (target substance). However, inhalation repeated dose toxicity data are available for sodium tungstate (source substance), which will be used for reading across. Due to lower water solubility and lower toxicity for the target substance compared to the source substance, the resulting read across from the source substance to the target substance is appropriate as a conservative estimate of potential toxicity for this endpoint. In addition, read across is appropriate because the classification and labelling is more protective for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2010-08-31 to 2010-01-29
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The reliability of this study for the substance tested is a K1, but in application of read-across to a different substance ECHA’s guidance specifies that the score can be a maximum of K2. Due to higher water solubility and greater in vitro bioaccessibility in synthetic alveolar, lysosomal, and interstitial fluids simulating inhalation exposure for the source substance (TBO) as compared to the target substance (tungsten carbide) and lack of toxicity from acute toxicity studies for the target and source substances, toxicity data on the source substance is expected to represent a worse case, so read-across is appropriate between these substances. In addition, read-across is appropriate for this endpoint because the classification and labelling for human health toxicity endpoints is the same for the source and target substances, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, sufficiently similar or lower for the source substance. For more details, refer to the attached description of the read-across approach.
Justification for type of information:
1. HYPOTHESIS FOR THE CATEGORY APPROACH: The hypothesis is that properties are likely to be similar or follow a similar pattern because of the presence of a common metal ion, in this case tungstate.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES):
Source: Tungsten Oxide
Target: RM W2C + WC
3. CATEGORY APPROACH JUSTIFICATION: See Annex 1 in CSR
4. DATA MATRIX: See Annex 1 in CSR
Reason / purpose:
reference to same study
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, (St. Constant, Canada)
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: One day following receipt, body weight ranges of the first shipment of rats 226 to 279 g (males) and 146 to 173 g (females). One day following receipt, body weight range of the second shipment of male rats was 207 to 234 g.
- Fasting period before study: no food or water was provided during exposures.
- Housing: At the start of food consumption measurements, the rats were individually housed in clear polycarbonate rodent cages (Allentown Caging Equipment Co., Allentown, NJ).
-Diet: Certified Rodent Chow 5002 meal (PMI Nutrition International, Inc., Brentwood, MO) was provided ad libitum, except during inhalation exposures and scheduled fasting periods. Diet analysis reports received from the supplier are maintained with facility records. The diet contained no known contaminants at levels that would be expected to interfere with the test substance or the animals or confound interpretation of the study.
- Water (e.g. ad libitum): Each rodent cage was provided with an automatic watering system (Edstrom Industries, Inc., Waterford, WI) supplying fresh city of Chicago water without additional treatment ad libitum, except during inhalation exposures.
- Acclimation period: The animals were quarantined for 2 weeks; To condition the animals for placement and restraint in the nose-only exposure tubes, and reduce stress during the exposure phase, the animals were acclimated to the restraining tubes during a three-day acclimation period. Animals were restrained for 1/4 (1.5 hours), 1/2 (3 hours), and 3/4 (4.5 hours) of the daily exposure duration (6 hours) on three non-holiday weekdays before the animals were exposed.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.6 to 23.0 degree C
- Humidity (%): 25.1-64.6%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): automatic 12-hour light/dark cycle was maintained in the exposure and housing chamber laboratories.



IN-LIFE DATES: From: 2010-09-09 To: 2010-10-21
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: The mean MMADs of the test atmosphere were 2.63, 2.87 and 2.74 um with GSDs of 1.89, 1.94 and 1.92 for Groups 2 through 4, respectively.

Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The nose-only chamber employed for test substance exposure was contained in an acrylic enclosure to isolate the exposure chamber and protect laboratory personnel. The dilution air to the atmosphere generator was of breathable quality and was filtered with a compressed air filter and a carbon absorber. The exhaust from the exposure chamber was moved through a particulate filter by a ring compressor and exhausted outside the building. Inlet and exhaust flows to and from the chamber were continuously monitored by rotameters.
- Method of holding animals in test chamber: During the inhalation exposures, the rats were restrained in nose-only exposure animal holding tubes (CH Technologies, Westwood, NJ). Animal tube loading and unloading, and tube insertion and removal from the exposure chamber were performed
according to standard procedures designed to minimize stress to study rats. At all times that rats were restrained in holders, they were observed
frequently and when necessary, action was taken to avoid injury, death, or improper exposure. Prior to the start of the exposure, rats were transferred from their housing cages to the nose-only holding tubes. Following confirmation of correct animal number, the animals in the holders were inserted into the ports of the exposure chambers. Following the exposure, the holders were removed. The rats were removed from the holders and returned to their home cages. Chamber port rotation occurred weekly.
- System of generating particulates/aerosols: Test atmospheres in the exposure chambers were generated by aerosolizing the test substance using a compressed air-operated Wright Dust Aerosol Generation System positioned over the chamber. Each inhalation exposure system was equipped with a separate aerosol generation system. The test substance was weighed out and packed into a dust reservoir daily. A constant speed rotating scraper separated a thin film of the test substance at the surface of the cake and delivered it into a dispersing unit, drawn in by aspiration and dispersed by a high velocity air jet. The resulting test atmosphere entered a mixing plenum where it was diluted with breathable quality compressed air to the target concentration prior to introduction to the nose-only inhalation exposure chamber.
- Air flow rate: The total airflow was set to produce an airflow range of approximately 0.5 to 1.0 L/min/exposure port.
- Method of particle size determination: The aerosol particle size distribution was monitored twice per week during the exposure phase of the study by an Aerodynamic Particle Sizer (APS) 3321 with Aerosol Diluter 3302A (both manufactured by TSI Inc., Shoreview, MN). The APS sizes particles in the range from 0.5 to 20 um using a time-of-flight technique that measures aerodynamic diameter in real time.


TEST ATMOSPHERE
- Brief description of analytical method used: The test atmosphere mass concentration was monitored gravimetrically by collecting gravimetric samples on pre-weighed glass fiber filters placed in closed-face filter holders. Samples were collected at a constant flow rate equal to the port flow of the delivery tube, and the total volume of air sampled was measured by a dry gas meter. Test atmosphere samples were collected at least three times during the exposure (generally, once during the first two hours, once during the middle two hours and once during the last two hours). The filter-collected samples were weighed and one filter per group per day (including the control to confirm the absence of test substance in the test atmosphere) was analyzed chemically to confirm the mass of TBO collected; percent recovery (chemical analysis concentration vs. gravimetric concentration) was calculated for each filter analyzed. Chemical analysis was conducted by means of ICP-mass spectrometry. In addition, the test atmosphere aerosol concentration in each chamber was monitored with a real-time aerosol sensor (model # pDR-1000AN, MIE, Inc. Bedford, MA). The sensors were employed only as a real-time indicator of short-term changes in aerosol concentration and were used in guiding laboratory personnel if concentration excursions were encountered.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
6 hours/day, 7 days/week for 28 days (14-day recovery period)
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0.08, 0.325 and 0.65 mg/L Air
Basis:
other: Target TBO Concentration
Remarks:
Doses / Concentrations:
15.2, 61.8, 123.6 mg/kg/day
Basis:
other: Target Inhaled Concentration (calculated)
Remarks:
Doses / Concentrations:
0.081, 0.331, and 0.652 mg/L
Basis:
other: mean concentrations determined gravimetrically
Remarks:
Doses / Concentrations:
14.8, 60.2, and 118.8 mg/kg/day
Basis:
other: mean inhaled concentrations (calculated)
No. of animals per sex per dose:
5 animals/sex/group
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment: Rats were assigned to groups using a computerized randomization procedure based on body weights using a "measure random" method that will produce similar group mean values.
- Group 1: Control
- Group 2: Low dose group
- Group 3: Mid dose group
- Group 4: High dose group
- Rationale for selecting satellite groups: Designated Subgroup
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The toxicology animals were observed for mortality and moribundity once daily during quarantine, twice daily during the exposure period (once in the morning and once in the afternoon) and once daily during the recovery period.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animal groups (core and recovery) received a thorough clinical examination daily during the study. Clinical observations during the exposure period were recorded before the exposure and within one hour after exposure termination following removal from the exposure chamber and holding tube. All clinical signs of altered behavior, changes in coat condition, unusual discharge of body fluid, abnormal respirations, lesions or other relevant findings were recorded. Clinical observations were recorded using ToxData© System, version 2.1.E.5 (Pathology Data Systems, Basel, Switzerland).


BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of all animals were determined one day after receipt and on the day of randomization to facilitate test subject selection. Body weights were measured for core and recovery animals on study days 1, 8, 15, 22, 28 (body weights for recovery animals were measured on study Day 29); fasted body weight measured on Day 29 (core animals only). During the recovery period, body weights were measured on study Days 36, 42 and a fasted body weight on study Day 43. Body weight measurements were collected using ToxData© System, version 2.1.E.5.


FOOD CONSUMPTION:
- Food consumption for each animal was determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; Food consumption for the core and recovery animals was measured five days prior to the first exposure (included in the study data but not reported), and daily on study Days 1, 8, 15, 22 and 29. During the recovery period, food consumption was measured on study Days 36 and 42. Food consumption was collected using ToxData© System, version 2.1.E.5.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Performed on all core animals on study Day 29 and all recovery animals on study Day 43.
- Anaesthetic used for blood collection: Yes; 70% CO2/30% O2
- Animals fasted: Yes
- How many animals: All animals
- Parameters examined: Red blood cell count and morphology, hematocrit, hemoglobin concentration, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, total and differential leukocyte count (absolute and relative), reticulocyte count (absolute
and relative) and platelet count. The following coagulation parameters were evaluated: fibrinogen, prothrombin time and activated partial thromboplastin time. The data was evaluated using a Diagnostica Stago STA Compact CT Coagulation Analyzer (DIAGNOSTICA STAGO, Inc., Parsippany, NJ), the Coagulation data were transferred from the Coagulation Analyzer to ToxData System, version 2.1.E.5.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Performed on all core animals on study Day 29 and all recovery animals on study Day 43.
- Animals fasted: Yes
- How many animals: All animals
- Parameter examined: alanine aminotransferase, albumin, alkaline phosphatase, aspartate aminotransferase, total bilirubin, blood urea nitrogen, calcium, chloride, cholesterol, creatinine, gamma-glutamyl transpeptidase, glucose, lactate dehydrogenase, phosphorus, potassium, sodium, total protein and triglycerides.


URINALYSIS: Yes
- Time schedule for collection of urine: Performed on all core animals on study Day 29 and all recovery animals on study Day 43.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: pH, protein, glucose, ketones, occult blood, bilirubin, urobilinogen, nitrite and leukocytes. Refractive index was measured with a refractometer, and a species-specific urine solids table was used to convert the refractive index to specific gravity. Urine was evaluated macroscopically for color, clarity and volume, and the sediment was analyzed microscopically.


OTHER:
ORGAN WEIGHTS: At the terminal and recovery necropsies, the adrenal glands, brain, epididymides, paired kidneys, liver, lungs, spleen, uterus, thymus and paired gonads (testes or ovaries) were removed, trimmed, and weighed. Organ weight/body weight ratios (relative organ weights) were calculated using the fasted body weights obtained prior to necropsy.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes; complete necropsies were conducted on all animals at terminal and recovery sacrifice. The necropsy included examination of the external surface and all orifices; all body cavities including the cranial cavity; and collection and fixation of the following tissues and organs: adrenal glands, brain, epididymides, heart (with aorta), kidneys, liver (two sections including left lateral and median lobes), lungs (with mainstem bronchi), ovaries, pancreas, seminal vesicles, spleen, sternum (bone marrow), testes, thymus and uterus. Gross lesions were also collected and preserved from all animals. In addition, the section of the tail bearing the animal identification number was also collected and preserved from all animals, and two bone marrow smears were collected from the femur of each animal.

HISTOPATHOLOGY: Yes; The adrenal glands, kidneys, liver (two sections including left lateral and median lobes), lungs (with mainstem bronchi), spleen and any gross lesions for the control and high dose groups were processed for microscopic evaluation for all core and recovery animals at necropsy (except as noted in Protocol Deviation No. 1). Tissues from other dose group animals were saved for future evaluations. Lungs (with mainstem bronchi) for the low and mid dose groups were processed for microscopic evaluation for all core toxicology animals at terminal necropsy.
Statistics:
Clinical observations were tabulated, but not statistically analyzed.

Body weight, body weight gain, food consumption and clinical pathology (haematology, coagulation and clinical chemistry) were analyzed for normality and equal variance. If the data set was normally distributed and of equal variance, statistical comparisons were conducted using a one-way analysis of variance (ANOVA), with post-hoc comparisons made using Dunnett's test. If normality and/or equal variance failed for a data set, statistical comparisons were conducted using the nonparametric Kruskal-Wallis ANOVA, with post-hoc comparisons made using Dunn's test. These parameters were compared using the statistical software provided by ToxData System, version 2.1.E.5. Urinalysis and organ weight data were compared using SYSTAT Software, version 10.2 (Systat Software Inc., Chicago, IL). Each sex was analyzed separately. Probability values of p < 0.05 (ToxData) or p 0.05 (SYSTAT) were considered significant.


Urinalysis (refractive index, specific gravity and pH only) and organ weight data were compared using ANOVA, with post-hoc comparisons made using Dunnett's test.

The Filtered Air Control group (Group 1) served as the control group for all comparisons.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
- No exposure-related deaths occurred during the study.
- The following clinical observations were observed during the pre- and post-exposure periods: skin/fur discoloration (blue), discoloration around the mouth, redness around nose fur, redness around the eyes, salivation, scab, injury, wet inguinal fur and limping. These observations (with the exception of skin/fur discoloration, discoloration around the mouth, scab, injury, and limping) are typical in nose-only exposure studies and were regarded as consequential to the rigors of nose-only exposure tube confinement.


BODY WEIGHT AND WEIGHT GAIN
- There were no statistically significant differences in group mean body weights for the male or female animals. Mean body weight gain was statistically significantly increased in High group females for Day 22-28, but the increase was not considered biologically relevant. No dose-related pattern was observed in either sex during the study.


FOOD CONSUMPTION
- There were no statistically significant differences in food consumption measurements, and no dose-related trend was observed in either sex.


HAEMATOLOGY
- At the end of the exposure period, white blood cells (WBC) and absolute eosinophils were statistically significantly increased in males in the Mid group, and absolute neutrophils, monocytes and eosinophils were statistically significantly increased in males in the High group, when compared to the Filtered Air Control. In females, relative reticulocytes were statistically significantly increased in the Mid group compared to the Filtered Air Control group. At the end of the recovery period, hemoglobin, hematocrit, MCV and absolute large unstained cells were statistically significantly increased in the High group males, when compared to the Filtered Air Control. At the end of the recovery period, absolute and relative reticulocytes were statistically significantly increased in the High group females, when compared to the Filtered Air Control. All red blood cell morphology observations were normal at the end of the exposure and recovery periods.
- Coagulation: At the end of the exposure period, no statistically significant differences were seen in males or females. No statistically significant differences were seen in males or females at the end of the recovery period.



CLINICAL CHEMISTRY
- At the end of the exposure period, calcium and glucose were statistically significantly increased in males in the Mid group and phosphorus was statistically significantly increased in Mid and High group males compared to the Filtered Air Control group. In females, gamma-glutamyl transpeptidase was significantly decreased in the Low group, calcium was statistically significantly increased in the Mid group, and chloride, phosphorus and albumin/globulin ratio levels were statistically significantly increased and globulin levels were statistically significantly decreased in the High group, compared to the Filtered Air Control group. At the end of the recovery period, cholesterol was statistically significantly increased in High group males, when compared to the Filtered Air Control. No statistically significant differences were seen in females at the end of the recovery period.


URINALYSIS
- Prior to terminal necropsy, urine refractive index and specific gravity were statistically significantly decreased for males in the low and High groups. At the end of the recovery period, no statistically significantly differences were observed between the groups.


ORGAN WEIGHTS
- At terminal necropsy, mean absolute and relative lung weights were statistically significantly increased in the Low, Mid and High groups for males, and in the Mid and High groups for females, compared to the Filtered Air Control group. At the recovery necropsy, mean absolute and relative lung weights were statistically significantly increased in males and females in the High group, mean relative testes weights were statistically significantly increased in males in the High group, and mean absolute and relative adrenal weights were statistically significantly decreased in males in the High group.


GROSS PATHOLOGY
- At Day 29, animals exposed to 0.65 mg/L air TBO had an increased incidence of pigmentation of the lungs which was correlated with the microscopic finding of alveolar foreign material and alveolar pigmented macrophages. Some animals exposed to 0.08 or 0.325 mg/L air levels also had pigmentation of the lungs with similar microscopic findings. In addition, the skin over the nasal bone was pigmented blue in all groups exposed to TBO. At Day 43, animals exposed to 0.65 mg/L air TBO had an increased incidence of pigmentation of the lungs which was correlated with the microscopic finding of alveolar foreign material and/or alveolar pigmented macrophages.


HISTOPATHOLOGY: NON-NEOPLASTIC
- At terminal sacrifice, there was an increase in the severity of alveolar pigmented macrophages, alveolar foreign material and individual alveolar foamy macrophages in animals exposed to all target concentrations of TBO. These three findings were considered related to exposure to TBO at all dose levels. In addition, at the 0.325 mg/L air and 0.65 mg/L air dose males there was an increase in the incidence of aggregates of foamy macrophages in the alveoli; this finding was also in the females exposed to 0.65 mg/L air concentration. There is a clear exposure response relationship in males for aggregates of macrophages in alveoli; this relationship is present, but less clear in the females. The finding of aggregated foamy macrophages was considered related to exposure of test substance at the 0.325 and 0.65 mg/L air concentrations in males and at the 0.65 mg/L air in females.

- After 14 days without exposure, there was the same incidence of animals with alveolar pigmented macrophages; however, the severity was slightly
decreased. There was a decrease in the severity of animals in Group 4 (0.65 mg/L air TBO) which had alveolar foreign material and individual alveolar foamy macrophages. There was a very small decrease in severity of foamy macrophage aggregates after 14 days without exposure. There was limited recovery of findings when compared to the terminal sacrifice animals.
Dose descriptor:
NOAEL
Effect level:
> 0.65 other: mg/L air (target)
Sex:
male/female
Basis for effect level:
other: No significant effects were observed at any dose
Dose descriptor:
NOAEL
Effect level:
> 0.652 mg/L air (analytical)
Sex:
male/female
Basis for effect level:
other: no significant effects were observed at any dose
Critical effects observed:
not specified

Overall means for TBO concentrations were determined gravimetrically to be 0.081, 0.331 and 0.652 mg/L for Groups 2 through 4, respectively. The TBO % recovery ranged from 100.83-102.38%.. Small amounts of TBO in the chemically-analyzed filters for the Filtered Air Control group were attributed to contamination during the filter analysis processing and/or the calibration curve. The Filtered Air Control group filter-collected mean gravimetric value was 0.000 mg/L. The particle size distribution of the test atmosphere was within the respirable range. The overall mean TBO inhaled dose levels were 14.8, 60.2 and 118.8 mg/kg/day for Groups 2 through 4, respectively. The overall mean male TBO inhaled dose levels were 13.7, 55.7 and 110.2 mg/kg/day for Groups 2 through 4, respectively. The overall mean female TBO inhaled dose levels were 15.8, 64.7 and 127.3 mg/kg/day for Groups 2 through 4, respectively. The male inhaled dose levels were 10-11% below the target levels for all groups, while the female inhaled dose levels were 3-5% above the target levels for all groups. Prior to exposure initiation, the homogeneity of the test atmosphere in each TBO exposure chamber was confirmed.

Conclusions:
The NOAEL was determined to be > 0.65 mg/L exposed by nose-only TBO inhalation for 6 hours per day, 7 days per week for 28 consecutive days.
Executive summary:

No inhalation repeated dose toxicity data of sufficient quality are available for the RM of W2C + WC (target substance). However, inhalation repeated dose toxicity data are available for tungsten oxide (source substance), which will be used for reading across. Due to similar water solubility and lower toxicity for the target substance compared to the source substance, the resulting read across from the source substance to the target substance is appropriate. In addition, read across is appropriate because the classification and labelling is similar for the source substance than the target substance, the PBT/vPvB profile is the same, and the dose descriptors are, or are expected to be, lower for the source substance. For more details, refer to the read-across category approach included in the Category section of this IUCLID submission and/or as an Annex in the CSR.

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

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Repeated Dose Toxicity - Oral Route:

No repeat dose toxicity data of sufficient quality were available for the RM of W2C + WC (fused carbide); however, data were available for sodium tungstate, which will be used for reading-across. The LOAEL and NOAEL from the 90-day oral toxicity study were deemed to be 125 mg/kg/day and 75 mg/kg/day, respectively, based on histopathological effects reported in the kidneys of the 125 and 200 mg/kg/day dose groups. The BMDL10 derived from this data was calculated to be 102 mg/kg/day. The cutoff range for a category 2 classification under CLP for a 90-day oral toxicity study is between 10 and 100 mg/kg/day. The LOAEL of 125 mg/kg/day identified from the repeat dose oral toxicity study was greater than 100 mg/kg/day. In addition, the benchmark dose (BMDL10) based on the data from the 90-day oral toxicity studies, and using the kidney as the target organ, was calculated to be 102 mg/kg/day. Because the LOAEL from the 90-day study as well as the calculated BMDL10 were greater than the 100 mg/kg/day category 2 cutoff level under CLP, then a classification is not warranted.

Repeated Dose Toxicity - Inhalation Route:

No repeat dose toxicity data of sufficient quality were available for the RM of W2C + WC (fused carbide); however, data were available for tungsten carbide and tungsten oxide, which will be used for reading-across. No effects were reported in the 90 -day inhalation toxicity study on WC on which a classification could be based and the only dose tested was well below the classification criteria. Therefore, the dose from the 90-day inhalation toxicity study was insufficient for determining classification. Therefore, the 28-day inhalation toxicity study on TBO was used for read across. The cutoff range for a category 2 classification under CLP for a 28-day inhalation toxicity study is between 0.06 and 0.6 mg/L. The NOAEL from the 28-day inhalation toxicity study on TBO was > 0.65 mg/L. Because the NOAEL from the 28-day study was greater than the 0.6 mg/L category 2 cutoff level, classification is not warranted.