Tungsten dioxide

Repeated dose toxicity: inhalation

Administrative data

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.

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

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

read-across: supporting information

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tungsten oxide

EC Number:

254-413-8

EC Name:

Tungsten oxide

Cas Number:

39318-18-8

Molecular formula:

WOn (n=2.99 to 2.90)

IUPAC Name:

oxotungsten

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Tungsten blue oxide (TBO)
- Physical state: dark blue heavy powder
- Analytical purity: 99.7-100%
- Storage condition of test material: stored at room temperature (approximately 15-30 degree C) in its original container

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, (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

Administration / exposure

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 h/d, 7 days/week for 28 d (14-d recovery period)

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.08, 0.325 and 0.65 mg/L Air
Basis:
other: Target TBO Concentration

Doses / concentrations 2

Remarks:

Doses / Concentrations:
15.2, 61.8, 123.6 mg/kg/d
Basis:
other: Target Inhaled Concentration (calculated)

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0.081, 0.331, and 0.652 mg/L
Basis:
other: mean concentrations determined gravimetrically

Doses / concentrations 4

Remarks:

Doses / Concentrations:
14.8, 60.2, and 118.8 mg/kg/d
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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

1

Key result

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

2

Key result

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

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Overall means for TBO (also known as tungsten blue oxide or tungsten oxide) 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.

Applicant's summary and conclusion

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 repeated dose inhalation toxicity data of sufficient quality are available for tungsten dioxide (target substance). However, repeated dose inhalation toxicity data are available for tungsten oxide (source substance), which will be used for read-across. Due to lower water solubility and similar 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 equally protective for the source and target substances, 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 attached description of the read-across approach.