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Description of key information

Studies via the oral and inhalation route are not available for vanadium dioxide, but for other vanadium substances. The rationale for read-across to vanadium dioxide including the limitations thereof is summarised below (see discussion). Of the limited effects noted following oral exposure of soluble vanadium substances, it appears most likely that effects on haematological parameters are the most consistently reported among a number of investigators.
Information on repeated dose toxicity following inhalation exposure to V2O5 is available in a NTP study (k_NTP 2002) with exposure of male and female rats and mice to V2O5 over 16-days, 3-months and 2-years. Pulmonary reactivity to vanadium pentoxide was also investigated following subchronic inhalation exposure in a non-human primate animal model. However, local effects on the respiratory tract are not considered relevant for vanadium dioxide. In a 14-d repeated-dose inhalation toxicity study, Sprague-Dawley rats were exposed to micronised vanadium trioxide powder via nose-only inhalation.Test-substance related mortality or any clinical signs of systemic toxicity were not observed at any exposure level. Body weights/body weight gain and food consumption were significantly decreased only at the highest exposure (0.25 mg/L) in males and females. In summary, based on the local effects in the respiratory tract of the test animals at the high exposure level (0.25 mg/L), the NOAEC was 0.02 mg/L (20 mg/m3). All effects on BAL parameters, lung weights and lung histopathology seen at the lower exposure levels were considered as adaptive responses to divanadium trioxide exposure, rather than an indication of local toxicity. This NOAEC translates into 22 mg VO2 /m3.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-10-19 to 2011-11-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This range-finding study was conducted according to the study protocol and its amendments, as well as all applicable IITRI Standard Operation Procedures (SOPs). The study is comparable to the OECD Technical Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study) with respect to the standards of a range-finding study, especially to test conditions, particle size distribution of aerosols, actual concentration measurements of the test substance, bronchoalveolar lavage, gross pathology and histopatholgy.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Principles of method if other than guideline:
This study was conducted to determine the potential toxicity of aerosolized vanadium trioxide powder to male and female Sprague-Dawley rats exposed to the test substance via nose-only inhalation. The rats were exposed at concentrations of 0, 0.0022, 0.022 and 0.25 mg/L air (analytical) for six hours per day, five days per week, over the course of two weeks (excluding weekend days; yielding a total of 10 exposures). Evaluated toxicological endpoints consisted of mortality/clinical observations, body weights, food consumption, pulmonary lavage parameters (cell numbers, differentials, total protein and lactate dehydrogenase), organ weights, gross necropsy and histopathology.
GLP compliance:
no
Remarks:
except for the analysis of vanadium trioxide in collection filters. This analysis was conducted under GLP.
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS - CRL:CD®(SD)IGS BR
- Source: Charles River Laboratories' Portage, MI, facility
- Age at study initiation: 55 days
- Weight at study initiation: males: 203 - 239 g; females: 184 - 215 g
- Fasting period: approximately 16-19 hours prior to necropsy
- Housing: during the quarantine period, animals of the same sex were double-housed in polycarbonate cages with hardwood bedding. After randomization, animals were single-housed in stainless steel cages suspended over absorbent paper cage boards.
- Diet (ad libitum, except during inhalation exposure): Certified Rodent Diet No. 5002 (PMI Nutrition International, Inc., Brentwood, MO)
- Water (ad libitum, except during inhalation exposure): City of Chicago water
- Acclimation period: 6 days
To condition the animals to placement and restraint in the nose-only holding tubes, the animals were placed in the holding tubes over three days prior to the start of exposure to the test substance according to the following schedule: one and a halfhours on Day -5; three hours on Day -2; and 4 hours and 30 minutes on Day -1.

The health certificate from the vendor showed positive representative colony results for Staphylococcus aureus and beta-haemolytic streptococci Group B (Streptococcus agalactiae); however, in general, neither of these organisms affects an animal's suitability for use in research. During quarantine the rats were observed daily for signs of disease or general unthriftiness. At the end of the quarantine period, the rats were examined by the testing facility veterinarian to ensure their suitability as test subjects.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-23°C
- Relative humidity: 32-64%
- Air changes: minimumof 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: The overall mean MMAD values and the GSD ranges were as follows:
0.0022 mg/L: MMAD = 1.92 ± 0.12 µm; GSD = 1.70 - 2.49
0.022 mg/L: MMAD = 2.02 ± 0.09 µm; GSD = 1.67 - 2.85
0.25 mg/L: MMAD = 2.25 ± 0.06 µm; GSD = 1.53 - 2.38
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus/ Method of holding animals in test chamber: each flow-past nose-only inhalation exposure chamber (Lab Products Inc., Seaford, DE) is comprised of 52 ports. The test atmosphere inlet and exhaust configurations provided a uniform and continuous stream of fresh test atmosphere to the animals undergoing exposure.
The animals were restrained in nose-only exposure animal holding tubes (CH Technologies, USA, Westwood, NJ). Each tube was placed in a predesignated port of the inhalation exposure chamber. Chamber ports were rotated for each exposure.

- System of generating particulates/aerosols: the test atmosphere was generated by aerosolization of the test substance using compressed air-operated Wright Dust Feeder Aerosol Generation systems (BGI Incorporated, Waltham, MA), which were positioned over each chamber. The test substance was weighed and packed into a dust reservoir using a 10 ton hydraulic shop press (TorinJacks, Ontario, CA), forming a cake. Each cake was compressed to 2 tons with the hydraulic shop press. 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, when necessary, was diluted with breathable quality compressed air to achieve target concentration prior to introduction to the exposure chamber.
Exhaust from the exposure chambers was moved through a high efficiency particulate air (HEPA) filter by a ring compressor and exhausted outside the building. Inlet and exhaust flows to and from the chamber were controlled and continuously monitored by rotometers.

- Temperature, humidity, airflow rate, oxygen: oxygen percentage was measured once during the exposure with an Altair Oxygen Sensor and Detector (MSA Instrument Division, Pittsburgh, PA) to ensure that a safe oxygen level was maintained for the animals.
Inhalation exposure chamber temperature, relative humidity and airflow rate (standard cubic feet per hour; SCFH) were measured and recorded at approximate 30-minute intervals during the exposure. The chamber temperature and relative humidity were monitored with a hand-held thermohygrometer (35612 series, Oakton Instruments, Vernon Hills, IL ).
Overall mean chamber temperatures were 21°C for all chambers, while overall mean relative humidity levels were 20 - 21% and overall mean oxygen levels were 21%. The mean relative humidity levels were below the targeted range of 30 - 70% due to the large volumes of dry compressed air needed to generate aerosol test atmospheres. Overall mean volumetric airflow rate data indicated that the test atmosphere was delivered effectively to the exposure chambers while maintaining safe oxygen levels for the animals.

- Method of particle size determination: aerosol particle size distribution was determined once during each exposure with a quartz crystal microbalance (QCM) cascade impactor (California Measurements Inc., Sierra Madre, CA) equipped with 10 stages to collect size-segregated samples. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were calculated from the mass accumulated on each collection stage of the QCM.

The chambers were encased in an acrylic enclosure to isolate the exposure chamber and protect laboratory personnel.

Prior to conducting the study, a validation of the exposure system was conducted to establish a suitable method for test atmosphere generation.

TEST ATMOSPHERE
- Brief description of analytical method used: the test atmosphere concentration in each exposure chamber was determined gravimetrically each exposure day by collecting test atmosphere samples on membrane filters placed in closed-face filter holders in the breathing zone of the animals. The gravimetric sampling train consisted of a preweighed 47 mm Teflon membrane filter (GE Water and Process Technologies, Westborough, MA) in series with a dry-gas meter connected to a constant flow vacuum pump. Samples were collected at a constant flow rate equal to the port flow of the delivery tube. The filter samples were weighed to determine the aerosol mass collected. The dry-gas meter measured the corresponding volume of chamber
air sampled and the weight-to-volume ratio was determined to obtain the aerosol mass concentration. Samples were collected at the following frequencies:
0 mg/L: 360 minutes (nominal sampling time); 1 sample/exposure
0.0022 mg/L: 360 minutes (nominal sampling time); 1 sample/exposure
0.022 mg/L: 120 minutes (nominal sampling time); 3 sample/exposure
0.25 mg/L: 30 minutes (nominal sampling time); 3 sample/exposure
One filter each from the 0.022 and 0.25 mg/L group Exposure/Study Day 1 exposures was analyzed by ICP-MS for determination of vanadium content and to confirm the gravimetric weight measurement. A filter from 0.0022 mg/L group was not analyzed by ICP-MS due to the small quantity of material collected on filters.
Test substance concentration values as determined by ICP-MS for one filter each from the 0.022 and 0.25 mg/L group Exposure/Study Day 1 exposures were 95% and 98% of gravimetric values for the two levels, respectively (please also refer to "Attached background material" below).
Aerosol concentration was monitored with a real-time aerosol sensor (model#pDR-1000AN, MIE, Inc. Bedford, MA). The sensors were employed as a realtime 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
Details on analytical verification of doses or concentrations:
Please refer to "Details on inhalation exposure" above.
Duration of treatment / exposure:
2 weeks (excluding weekend days; yielding a total of 10 exposures)
Frequency of treatment:
6 hours per day, 5 days per week
Remarks:
Doses / Concentrations:
0.0022 mg/L
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
0.022 mg/L
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
0.25 mg/L
Basis:
analytical conc.
No. of animals per sex per dose:
6 males / 6 females
Control animals:
yes, concurrent vehicle
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations: mortality, moribundity and general appearance

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before and after (within two hours) exposure
Examinations included observations of general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia, as well as evaluations of respiration and behavior.

Beginning on Study Day 2 (Study day 1 is Exposure Day 1), the nose area of the test substance-exposed animals was wiped with a wet paper towel to remove excess test substance; this was done postexposure, but prior to and/or in conjunction with clinical observations.

BODY WEIGHT: Yes
- Time schedule for examinations: one day after animal receipt; at randomization; and prior to exposure on Days 1, 8, 14, and 15.
A final (fasted) body weight was collected on Study Day 15 for each animal (one day following the final exposure).
Body weight changes were calculated for all rats.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
Food consumption measurements were collected on Days 1, 8 and 14 (concurrently with body weights) and calculated for the Days 1-8 and 8-14 intervals.

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

PULMONARY LAVAGE:
Bronchial alveolar lavage (BAL) was performed on Study Day 15 (after 10 exposures) on the left lobe of the lung of all study animals.
BAL fluid was analysed for the following: lactate dehydrogenase (LDH), protein analysis, cell counts (concentration, cells/lung, viable cells/lung and percent viable cells) and differential counts (segmented neutrophils, lymphocytes, monocytes/macrophages, eosinophils, and basophils)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Terminal fasted body weights were recorded on Study Day 15 (one day after the last exposure). The animals were euthanized by sodium pentobarbital followed by exsanguination from the abdominal aorta and were given a complete necropsy on Study Day 15.

At necropsy, the external surface of the body, all orifices, and the cranial, thoracic and peritoneal cavities and their contents were examined and any lesions or abnormal conditions (gross pathologic fmdings) were recorded. Selected organs were weighed (lung, liver, kidneys, adrenals, brain, spleen and ovaries or testes) and organ-to-body weight ratios were calculated using the terminal (fasted) body weight for each animal. The following tissues were collected and fixed in 10% neutral buffered formalin: bronchi, lung (right), bronchial lymph node, trachea, gross lesions and target tissues (mediastinal lymph node and thymus). All livers were also retained in formalin because a gross lesion in the liver was observed for the first 0.25 mg/L group animal necropsied (male animal); however, no gross lesions in the liver were observed for any other 0.25 mg/L group animals and thus the liver was not considered a target tissue.

HISTOPATHOLOGY: Yes
Tissue samples were trimmed, processed, embedded in paraffin, sectioned at approximately 5 µm, mounted on slides, and stained with hematoxylin
and eosin for the groups as follows:
- Air control group and 0.25 mg/L group males and females - bronchi, lung (right), bronchial lymph node, trachea, mediastinal lymph node, thymus and gross lesions
- 0.002 mg/L group and 0.02 mg/L group males and females - tissues identified as target tissues; e.g., lung, bronchial lymph node, mediastinal lymph node and thymus
Histopathological findings were recorded.
Statistics:
Descriptive statistics (mean and standard deviation) were calculated for data in the following categories: test atmosphere, body weight/body weight change,
food consumption, pulmonary lavage, and organ weight/organ-to-body weight ratios. The continuous data in the above categories, with the exception of test
atmosphere, were analyzed for statistical significance. A minimum significance level of p ≤ 0.05 was used for the comparisons.
If the data sets were normally distributed and of equal variance, statistical comparisons were conducted using one-way analysis ofvariance (ANOVA), with post hoc comparisons (if necessary) made using Dunnett's test. If normality and/or equal variance tests failed for a data set, statistical comparisons were conducted using nonparametric Kruskal-Wallis ANOVA followed (if necessary) by Dunn's test.
Body weight/body weight change, food consumption, the pulmonary lavage parameter ofLDH, and organ weight/organ-to-body weight ratios were compared
using ToxData® (version 2.1.E). The remaining pulmonary lavage parameters (cell numbers, differentials and total protein concentration) were compared using
SigmaStat® Software, version 2.03 (Systat Software Inc., Chicago, IL).
The differentials category ofbasophils (pulmonary lavage) was not analyzed for statistical significance because all results were "0" for all animals ofboth sexes in all groups.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
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 test-substance related mortality or clinical signs of systemic toxicity were observed.

BODY WEIGHT AND WEIGHT GAIN
Statistically significant decreases in body weight in comparison to control group were observed for 0.25 mg/L group males and females on Day 8 (14 and 10%, respectively) and on Day 14 (24 and 17%, respectively), while statistically significant decreases in body weight change were observed for 0.25 mg/L group males and females for the Study Days 1-8, 8-14 and 1-14 intervals. Body weights were also slightly decreased in 0.022 mg/L group males on Day 8 (3.5% decrease in body weight) and on Day 14 ( 4.4% decrease in body weight); however, the decreases were not statistically significant.

FOOD CONSUMPTION
Statistically significant decreases in food consumption were observed for 0.25 mg/L group males and females for the Study Days 1-8 and 8-14 intervals. Food consumption was also slightly decreased in 0.022 mg/L group males at both of these intervals; however, the decreases were not statistically significant.

ORGAN WEIGHTS
The following statistically significant differences in organ weight data in comparison to control group were observed for the test-item treated groups:
1) Males
Absolute organ weights
0.022 mg/L:
- Increase: lung
0.25 mg/L:
- Increase: lung
- Decrease: kidneys and spleen

Relative organ weights (Organ-to-Body Weight Ratios)
0.022 mg/L:
- Increase: lung
0.25 mg/L:
- Increase: brain, lung, and testes
- Decrease: fasted body weight and spleen

2) Females - Absolute organ weights
0.022 mg/L:
- Increase: lung
0.25 mg/L:
- Increase: lung
- Decrease: spleen

Relative organ weights (Organ-to-Body Weight Ratios)
0.022 mg/L:
- Increase: lung
0.25 mg/L:
- Increase: brain, lung, and liver
- Decrease: fasted body weight

Absolute and relative lung weights were also increased in 0.0022 mg/L group males and females; however, the increases were not statistically significant. The increased lung weight seen in all groups and the decreased spleen weight seen in the 0.25 mg/L group males and females were considered exposure-related. The decreased absolute kidney weight in the 0.25 mg/L group males was not considered exposure-related since the relative weight was not decreased in this group. The increased relative brain, testes and liver weights were the result of the decreased fasted body weights, rather than an indication of direct organ toxicity.

GROSS PATHOLOGY
Test substance-related findings are summarized as follows (findings observed in males and females unless otherwise noted):
- 0.0022 mg/L group: dark pigmentation lung (females only) and enlarged bronchial lymph node
- 0.022 mg/L group: dark pigmentation lung; enlarged bronchial lymph node; enlarged mediastinal lymph node
- 0.25 mg/L group: dark pigmentation lung; enlarged and dark pigmentation bronchial lymph node; enlarged and dark pigmentation mediastinal lymph node; small thymus (males only)

HISTOPATHOLOGY: NON-NEOPLASTIC
Test substance-related findings are summarized as follows (findings observed in males and females unless otherwise noted):
- 0.0022 mg/L group:
Lung: pigmentation of marcophages in the alveolus; histiocytosis
Bronchial lymph node: hyperplasia of the paracortical zone (males only)
- 0.022 mg/L group:
Lung: pigmentation of marcophages in the alveolus
Bronchial lymph node: hyperplasia of the paracortical zone
Mediastinal lymph node: hyperplasia of the paracortical zone
- 0.25 mg/L group:
Lung: infiltration of mixed cells in the alveolus and interstitium; pigmentation of macrophages in the alveolus
Bronchial lymph node: hyperplasia of the paracortical zone
Mediastinal lymph node: hyperplasia of the paracortical zone
Thymus: atrophy (males only)

PULMONARY LAVAGE:
The following statistically significant differences in pulmonary lavage data comparison to the control group were observed for the test-item treated groups:
1) Males
0.0022 mg/L group:
- Increase: segmented neutrophils (2-fold), monocytes/macrophages (2-3 fold), total protein, and LDH
- Decrease: lymphocytes
0.022 mg/L group:
- Increase: cell concentration, cells/lung, viable cells/lung, monocytes/macrophages (2-3 fold), total protein, and LDH
- Decrease: lymphocytes
0.25 mg/L group:
- Increase: segmented neutrophils (2-fold), monocytes/macrophages (2-3 fold), total protein, and LDH
- Decrease: lymphocytes
2) Females
0.0022 mg/L group:
- Increase: cells/lung, viable cells/lung, and monocytes/macrophages (2-3 fold)
- Decrease: lymphocytes
0.022 mg/L group:
- Increase: cell concentration, cells/lung, viable cells/lung, monocytes/macrophages (2-3 fold), total protein, and LDH
- Decrease: lymphocytes
0.25 mg/L group:
- Increase: cell concentration, cells/lung, viable cells/lung, segmented neutrophils(2-fold), monocytes/macrophages (2-3 fold), total protein, and LDH
- Decrease: lymphocytes

Increases, although not statistically significant, also were noted in cell concentration in 0.0022 mg/L group males and females; cells/lung and viable cells/lung in 0.0022 mg/L group males; protein concentration in 0.0022 mg/L group females; and LDH in 0.0022 mg/L group females.
For most of the BAL parameters, there was an exposure concentration response for the low and mid exposure groups; however, the values for the high exposure group were lower than those for the mid group. This may have been the result of the inability to fully recover the infused lavage fluid (possibly due to the lung being filled with particulate vanadium, as evidenced microscopically by pigmentation of alveolar macrophages, thus preventing removal/collection of the cells filling the alveoli).
No eosinophils and basophils were detected in the BAL fluid of all animals of both sexes in all groups including controls.
Dose descriptor:
NOAEC
Effect level:
0.022 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOAEL is based on decreased body weight at the high exposure level (0.25 mg/L air (analytical)).
Dose descriptor:
LOAEC
Effect level:
0.25 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Critical effects observed:
not specified
Conclusions:
The no-observed-adverse-effect concentration (NOAEC) in this study is established at the exposure concentration of 0.02 mg/L based on decreased body weights at the high exposure level of 0.25 mg/L (LOAEC). The changes of BAL parameters, lung weights and lung histopathology seen at all exposure levels can be considered adaptive responses to the exposure to vanadium trioxide aerosols.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-10-19 to 2011-11-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This range-finding study was conducted according to the study protocol and its amendments, as well as all applicable IITRI Standard Operation Procedures (SOPs). The study is comparable to the OECD Technical Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study) with respect to the standards of a range-finding study, especially to test conditions, particle size distribution of aerosols, actual concentration measurements of the test substance, bronchoalveolar lavage, gross pathology and histopatholgy.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Principles of method if other than guideline:
This study was conducted to determine the potential toxicity of aerosolized vanadium trioxide powder to male and female Sprague-Dawley rats exposed to the test substance via nose-only inhalation. The rats were exposed at concentrations of 0, 0.0022, 0.022 and 0.25 mg/L air (analytical) for six hours per day, five days per week, over the course of two weeks (excluding weekend days; yielding a total of 10 exposures). Evaluated toxicological endpoints consisted of mortality/clinical observations, body weights, food consumption, pulmonary lavage parameters (cell numbers, differentials, total protein and lactate dehydrogenase), organ weights, gross necropsy and histopathology.
GLP compliance:
no
Remarks:
except for the analysis of vanadium trioxide in collection filters. This analysis was conducted under GLP.
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS - CRL:CD®(SD)IGS BR
- Source: Charles River Laboratories' Portage, MI, facility
- Age at study initiation: 55 days
- Weight at study initiation: males: 203 - 239 g; females: 184 - 215 g
- Fasting period: approximately 16-19 hours prior to necropsy
- Housing: during the quarantine period, animals of the same sex were double-housed in polycarbonate cages with hardwood bedding. After randomization, animals were single-housed in stainless steel cages suspended over absorbent paper cage boards.
- Diet (ad libitum, except during inhalation exposure): Certified Rodent Diet No. 5002 (PMI Nutrition International, Inc., Brentwood, MO)
- Water (ad libitum, except during inhalation exposure): City of Chicago water
- Acclimation period: 6 days
To condition the animals to placement and restraint in the nose-only holding tubes, the animals were placed in the holding tubes over three days prior to the start of exposure to the test substance according to the following schedule: one and a halfhours on Day -5; three hours on Day -2; and 4 hours and 30 minutes on Day -1.

The health certificate from the vendor showed positive representative colony results for Staphylococcus aureus and beta-haemolytic streptococci Group B (Streptococcus agalactiae); however, in general, neither of these organisms affects an animal's suitability for use in research. During quarantine the rats were observed daily for signs of disease or general unthriftiness. At the end of the quarantine period, the rats were examined by the testing facility veterinarian to ensure their suitability as test subjects.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-23°C
- Relative humidity: 32-64%
- Air changes: minimumof 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: The overall mean MMAD values and the GSD ranges were as follows:
0.0022 mg/L: MMAD = 1.92 ± 0.12 µm; GSD = 1.70 - 2.49
0.022 mg/L: MMAD = 2.02 ± 0.09 µm; GSD = 1.67 - 2.85
0.25 mg/L: MMAD = 2.25 ± 0.06 µm; GSD = 1.53 - 2.38
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus/ Method of holding animals in test chamber: each flow-past nose-only inhalation exposure chamber (Lab Products Inc., Seaford, DE) is comprised of 52 ports. The test atmosphere inlet and exhaust configurations provided a uniform and continuous stream of fresh test atmosphere to the animals undergoing exposure.
The animals were restrained in nose-only exposure animal holding tubes (CH Technologies, USA, Westwood, NJ). Each tube was placed in a predesignated port of the inhalation exposure chamber. Chamber ports were rotated for each exposure.

- System of generating particulates/aerosols: the test atmosphere was generated by aerosolization of the test substance using compressed air-operated Wright Dust Feeder Aerosol Generation systems (BGI Incorporated, Waltham, MA), which were positioned over each chamber. The test substance was weighed and packed into a dust reservoir using a 10 ton hydraulic shop press (TorinJacks, Ontario, CA), forming a cake. Each cake was compressed to 2 tons with the hydraulic shop press. 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, when necessary, was diluted with breathable quality compressed air to achieve target concentration prior to introduction to the exposure chamber.
Exhaust from the exposure chambers was moved through a high efficiency particulate air (HEPA) filter by a ring compressor and exhausted outside the building. Inlet and exhaust flows to and from the chamber were controlled and continuously monitored by rotometers.

- Temperature, humidity, airflow rate, oxygen: oxygen percentage was measured once during the exposure with an Altair Oxygen Sensor and Detector (MSA Instrument Division, Pittsburgh, PA) to ensure that a safe oxygen level was maintained for the animals.
Inhalation exposure chamber temperature, relative humidity and airflow rate (standard cubic feet per hour; SCFH) were measured and recorded at approximate 30-minute intervals during the exposure. The chamber temperature and relative humidity were monitored with a hand-held thermohygrometer (35612 series, Oakton Instruments, Vernon Hills, IL ).
Overall mean chamber temperatures were 21°C for all chambers, while overall mean relative humidity levels were 20 - 21% and overall mean oxygen levels were 21%. The mean relative humidity levels were below the targeted range of 30 - 70% due to the large volumes of dry compressed air needed to generate aerosol test atmospheres. Overall mean volumetric airflow rate data indicated that the test atmosphere was delivered effectively to the exposure chambers while maintaining safe oxygen levels for the animals.

- Method of particle size determination: aerosol particle size distribution was determined once during each exposure with a quartz crystal microbalance (QCM) cascade impactor (California Measurements Inc., Sierra Madre, CA) equipped with 10 stages to collect size-segregated samples. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were calculated from the mass accumulated on each collection stage of the QCM.

The chambers were encased in an acrylic enclosure to isolate the exposure chamber and protect laboratory personnel.

Prior to conducting the study, a validation of the exposure system was conducted to establish a suitable method for test atmosphere generation.

TEST ATMOSPHERE
- Brief description of analytical method used: the test atmosphere concentration in each exposure chamber was determined gravimetrically each exposure day by collecting test atmosphere samples on membrane filters placed in closed-face filter holders in the breathing zone of the animals. The gravimetric sampling train consisted of a preweighed 47 mm Teflon membrane filter (GE Water and Process Technologies, Westborough, MA) in series with a dry-gas meter connected to a constant flow vacuum pump. Samples were collected at a constant flow rate equal to the port flow of the delivery tube. The filter samples were weighed to determine the aerosol mass collected. The dry-gas meter measured the corresponding volume of chamber
air sampled and the weight-to-volume ratio was determined to obtain the aerosol mass concentration. Samples were collected at the following frequencies:
0 mg/L: 360 minutes (nominal sampling time); 1 sample/exposure
0.0022 mg/L: 360 minutes (nominal sampling time); 1 sample/exposure
0.022 mg/L: 120 minutes (nominal sampling time); 3 sample/exposure
0.25 mg/L: 30 minutes (nominal sampling time); 3 sample/exposure
One filter each from the 0.022 and 0.25 mg/L group Exposure/Study Day 1 exposures was analyzed by ICP-MS for determination of vanadium content and to confirm the gravimetric weight measurement. A filter from 0.0022 mg/L group was not analyzed by ICP-MS due to the small quantity of material collected on filters.
Test substance concentration values as determined by ICP-MS for one filter each from the 0.022 and 0.25 mg/L group Exposure/Study Day 1 exposures were 95% and 98% of gravimetric values for the two levels, respectively (please also refer to "Attached background material" below).
Aerosol concentration was monitored with a real-time aerosol sensor (model#pDR-1000AN, MIE, Inc. Bedford, MA). The sensors were employed as a realtime 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
Details on analytical verification of doses or concentrations:
Please refer to "Details on inhalation exposure" above.
Duration of treatment / exposure:
2 weeks (excluding weekend days; yielding a total of 10 exposures)
Frequency of treatment:
6 hours per day, 5 days per week
Remarks:
Doses / Concentrations:
0.0022 mg/L
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
0.022 mg/L
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
0.25 mg/L
Basis:
analytical conc.
No. of animals per sex per dose:
6 males / 6 females
Control animals:
yes, concurrent vehicle
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations: mortality, moribundity and general appearance

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before and after (within two hours) exposure
Examinations included observations of general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia, as well as evaluations of respiration and behavior.

Beginning on Study Day 2 (Study day 1 is Exposure Day 1), the nose area of the test substance-exposed animals was wiped with a wet paper towel to remove excess test substance; this was done postexposure, but prior to and/or in conjunction with clinical observations.

BODY WEIGHT: Yes
- Time schedule for examinations: one day after animal receipt; at randomization; and prior to exposure on Days 1, 8, 14, and 15.
A final (fasted) body weight was collected on Study Day 15 for each animal (one day following the final exposure).
Body weight changes were calculated for all rats.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
Food consumption measurements were collected on Days 1, 8 and 14 (concurrently with body weights) and calculated for the Days 1-8 and 8-14 intervals.

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

PULMONARY LAVAGE:
Bronchial alveolar lavage (BAL) was performed on Study Day 15 (after 10 exposures) on the left lobe of the lung of all study animals.
BAL fluid was analysed for the following: lactate dehydrogenase (LDH), protein analysis, cell counts (concentration, cells/lung, viable cells/lung and percent viable cells) and differential counts (segmented neutrophils, lymphocytes, monocytes/macrophages, eosinophils, and basophils)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Terminal fasted body weights were recorded on Study Day 15 (one day after the last exposure). The animals were euthanized by sodium pentobarbital followed by exsanguination from the abdominal aorta and were given a complete necropsy on Study Day 15.

At necropsy, the external surface of the body, all orifices, and the cranial, thoracic and peritoneal cavities and their contents were examined and any lesions or abnormal conditions (gross pathologic fmdings) were recorded. Selected organs were weighed (lung, liver, kidneys, adrenals, brain, spleen and ovaries or testes) and organ-to-body weight ratios were calculated using the terminal (fasted) body weight for each animal. The following tissues were collected and fixed in 10% neutral buffered formalin: bronchi, lung (right), bronchial lymph node, trachea, gross lesions and target tissues (mediastinal lymph node and thymus). All livers were also retained in formalin because a gross lesion in the liver was observed for the first 0.25 mg/L group animal necropsied (male animal); however, no gross lesions in the liver were observed for any other 0.25 mg/L group animals and thus the liver was not considered a target tissue.

HISTOPATHOLOGY: Yes
Tissue samples were trimmed, processed, embedded in paraffin, sectioned at approximately 5 µm, mounted on slides, and stained with hematoxylin
and eosin for the groups as follows:
- Air control group and 0.25 mg/L group males and females - bronchi, lung (right), bronchial lymph node, trachea, mediastinal lymph node, thymus and gross lesions
- 0.002 mg/L group and 0.02 mg/L group males and females - tissues identified as target tissues; e.g., lung, bronchial lymph node, mediastinal lymph node and thymus
Histopathological findings were recorded.
Statistics:
Descriptive statistics (mean and standard deviation) were calculated for data in the following categories: test atmosphere, body weight/body weight change,
food consumption, pulmonary lavage, and organ weight/organ-to-body weight ratios. The continuous data in the above categories, with the exception of test
atmosphere, were analyzed for statistical significance. A minimum significance level of p ≤ 0.05 was used for the comparisons.
If the data sets were normally distributed and of equal variance, statistical comparisons were conducted using one-way analysis ofvariance (ANOVA), with post hoc comparisons (if necessary) made using Dunnett's test. If normality and/or equal variance tests failed for a data set, statistical comparisons were conducted using nonparametric Kruskal-Wallis ANOVA followed (if necessary) by Dunn's test.
Body weight/body weight change, food consumption, the pulmonary lavage parameter ofLDH, and organ weight/organ-to-body weight ratios were compared
using ToxData® (version 2.1.E). The remaining pulmonary lavage parameters (cell numbers, differentials and total protein concentration) were compared using
SigmaStat® Software, version 2.03 (Systat Software Inc., Chicago, IL).
The differentials category ofbasophils (pulmonary lavage) was not analyzed for statistical significance because all results were "0" for all animals ofboth sexes in all groups.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
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 test-substance related mortality or clinical signs of systemic toxicity were observed.

BODY WEIGHT AND WEIGHT GAIN
Statistically significant decreases in body weight in comparison to control group were observed for 0.25 mg/L group males and females on Day 8 (14 and 10%, respectively) and on Day 14 (24 and 17%, respectively), while statistically significant decreases in body weight change were observed for 0.25 mg/L group males and females for the Study Days 1-8, 8-14 and 1-14 intervals. Body weights were also slightly decreased in 0.022 mg/L group males on Day 8 (3.5% decrease in body weight) and on Day 14 ( 4.4% decrease in body weight); however, the decreases were not statistically significant.

FOOD CONSUMPTION
Statistically significant decreases in food consumption were observed for 0.25 mg/L group males and females for the Study Days 1-8 and 8-14 intervals. Food consumption was also slightly decreased in 0.022 mg/L group males at both of these intervals; however, the decreases were not statistically significant.

ORGAN WEIGHTS
The following statistically significant differences in organ weight data in comparison to control group were observed for the test-item treated groups:
1) Males
Absolute organ weights
0.022 mg/L:
- Increase: lung
0.25 mg/L:
- Increase: lung
- Decrease: kidneys and spleen

Relative organ weights (Organ-to-Body Weight Ratios)
0.022 mg/L:
- Increase: lung
0.25 mg/L:
- Increase: brain, lung, and testes
- Decrease: fasted body weight and spleen

2) Females - Absolute organ weights
0.022 mg/L:
- Increase: lung
0.25 mg/L:
- Increase: lung
- Decrease: spleen

Relative organ weights (Organ-to-Body Weight Ratios)
0.022 mg/L:
- Increase: lung
0.25 mg/L:
- Increase: brain, lung, and liver
- Decrease: fasted body weight

Absolute and relative lung weights were also increased in 0.0022 mg/L group males and females; however, the increases were not statistically significant. The increased lung weight seen in all groups and the decreased spleen weight seen in the 0.25 mg/L group males and females were considered exposure-related. The decreased absolute kidney weight in the 0.25 mg/L group males was not considered exposure-related since the relative weight was not decreased in this group. The increased relative brain, testes and liver weights were the result of the decreased fasted body weights, rather than an indication of direct organ toxicity.

GROSS PATHOLOGY
Test substance-related findings are summarized as follows (findings observed in males and females unless otherwise noted):
- 0.0022 mg/L group: dark pigmentation lung (females only) and enlarged bronchial lymph node
- 0.022 mg/L group: dark pigmentation lung; enlarged bronchial lymph node; enlarged mediastinal lymph node
- 0.25 mg/L group: dark pigmentation lung; enlarged and dark pigmentation bronchial lymph node; enlarged and dark pigmentation mediastinal lymph node; small thymus (males only)

HISTOPATHOLOGY: NON-NEOPLASTIC
Test substance-related findings are summarized as follows (findings observed in males and females unless otherwise noted):
- 0.0022 mg/L group:
Lung: pigmentation of marcophages in the alveolus; histiocytosis
Bronchial lymph node: hyperplasia of the paracortical zone (males only)
- 0.022 mg/L group:
Lung: pigmentation of marcophages in the alveolus
Bronchial lymph node: hyperplasia of the paracortical zone
Mediastinal lymph node: hyperplasia of the paracortical zone
- 0.25 mg/L group:
Lung: infiltration of mixed cells in the alveolus and interstitium; pigmentation of macrophages in the alveolus
Bronchial lymph node: hyperplasia of the paracortical zone
Mediastinal lymph node: hyperplasia of the paracortical zone
Thymus: atrophy (males only)

PULMONARY LAVAGE:
The following statistically significant differences in pulmonary lavage data comparison to the control group were observed for the test-item treated groups:
1) Males
0.0022 mg/L group:
- Increase: segmented neutrophils (2-fold), monocytes/macrophages (2-3 fold), total protein, and LDH
- Decrease: lymphocytes
0.022 mg/L group:
- Increase: cell concentration, cells/lung, viable cells/lung, monocytes/macrophages (2-3 fold), total protein, and LDH
- Decrease: lymphocytes
0.25 mg/L group:
- Increase: segmented neutrophils (2-fold), monocytes/macrophages (2-3 fold), total protein, and LDH
- Decrease: lymphocytes
2) Females
0.0022 mg/L group:
- Increase: cells/lung, viable cells/lung, and monocytes/macrophages (2-3 fold)
- Decrease: lymphocytes
0.022 mg/L group:
- Increase: cell concentration, cells/lung, viable cells/lung, monocytes/macrophages (2-3 fold), total protein, and LDH
- Decrease: lymphocytes
0.25 mg/L group:
- Increase: cell concentration, cells/lung, viable cells/lung, segmented neutrophils(2-fold), monocytes/macrophages (2-3 fold), total protein, and LDH
- Decrease: lymphocytes

Increases, although not statistically significant, also were noted in cell concentration in 0.0022 mg/L group males and females; cells/lung and viable cells/lung in 0.0022 mg/L group males; protein concentration in 0.0022 mg/L group females; and LDH in 0.0022 mg/L group females.
For most of the BAL parameters, there was an exposure concentration response for the low and mid exposure groups; however, the values for the high exposure group were lower than those for the mid group. This may have been the result of the inability to fully recover the infused lavage fluid (possibly due to the lung being filled with particulate vanadium, as evidenced microscopically by pigmentation of alveolar macrophages, thus preventing removal/collection of the cells filling the alveoli).
No eosinophils and basophils were detected in the BAL fluid of all animals of both sexes in all groups including controls.
Dose descriptor:
NOAEC
Effect level:
0.022 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOAEL is based on decreased body weight at the high exposure level (0.25 mg/L air (analytical)).
Dose descriptor:
LOAEC
Effect level:
0.25 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Critical effects observed:
not specified
Conclusions:
The no-observed-adverse-effect concentration (NOAEC) in this study is established at the exposure concentration of 0.02 mg/L based on decreased body weights at the high exposure level of 0.25 mg/L (LOAEC). The changes of BAL parameters, lung weights and lung histopathology seen at all exposure levels can be considered adaptive responses to the exposure to vanadium trioxide aerosols.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
22 mg/m³
Study duration:
subacute
Species:
rat

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: dermal
Data waiving:
other justification
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: dermal
Data waiving:
other justification
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Studies via the oral and inhalation route are not available for vanadium dioxide, but for other vanadium substances. The rationale for read-across to vanadium dioxide including the limitations thereof can be summarised according to the following relevant routes of exposure:

Read-across:

Unrestricted read-across from very soluble pentavalent substances to VO2 is not applicable since the bioaccessibility of VO2 is lower (and similar to V2O3 in comparison): in artificial gastric fluid at a loading of 100 mg/L V2O3, only 13% and 15% went into solution after 2h and 24h respectively, whereas other pentavalent substances (V2O5or NaVO3) dissolved completely within 2 h. A similarly low dissolution of V2O3was also observed in artificial lysosomal fluid (11.9% after 2h; 15.7% after 24h) and lung fluid (4.7% after 2h; 5.6% after 24h). Similar bioaccessibility tests are ongoing with VO2. For the time being, the solubility of V2O3 and VO2 in physiological fluids is expected to be similar based on similar intrinsic water solubility and transformation/dissolution characteristics. Thus, the biological uptake of vanadium from V2O3and from VO2 via the oral route is expected to be an order of magnitude lower compared to very soluble V substances, such as V2O5or NaVO3. Furthermore, the bioavailability and reactivity of V2O3in tissues of the respiratory tract is assumed to be far less than that of V2O5.Thus, any read-across from very soluble pentavalent vanadium substances to poorly/less soluble and poorly/less bioavailable for the purpose of classification and labelling of VO2 is not supported.

Oral:

A number of studies are available where vanadium compounds were administered, however they have involved different experimental approaches and designs as well as different dose regimens, and endpoints. The most consistent effect of exposure to V2O5, i.e. pulmonary irritation and inflammation, is associated with the inhalation route. For oral exposure to vanadium substances, effects are more limited and the different experimental approaches lead to a variety of endpoints measured. Of the limited effects noted following oral exposure, it appears most likely that effects on haematological parameters are the most consistently reported among a number of investigators (Mountain et al 1953, Zaporowska et al. 1993, Scibior et al 2006, Scibior, 2005, NTP, 2002).

 

In a study (treatment of male rats for 103 days) with the focus on reduction of the cysteine content in rat hair (Mountain et al. 1953), reduced erythrocyte counts and levels of hair cysteine were observed dose-dependently at dose levels of 100 and 150 ppm in the diet. Effects on hair cysteine levels and on red blood cell parameters may correlate with erythropenia and anaemia. Similar haematological effects were observed by Zaporowska et al. (1993) in a 4-week toxicity study. Rats received NH4VO3at dose levels of 1.5 and 5-6 mg V/kg bw/d via drinking water.

 

Haematological examinations showed a decrease in erythrocyte counts (associated with increased reticulocyte counts), haemoglobin and haematocrit levels in both groups. Effects of vanadium and chromium on body weight gain and selected haematological and blood parameters in rats were also investigated by Scibior (2005) following administration of NaVO3to rats via drinking water for a period of 6 weeks. Treatment of rats with about 8 mg V/kg bw/d resulted in effects on body weight and erythrocytes (increased no. of erythrocytes, haemoglobin, and decrease of MCV, MCH, MCHC and leucocytes).

 

Altogether, effects noted have included reduced haemoglobin, reduced haematocrit, reduced mean cell haemoglobin concentrations, while effects on red blood cells have included both reductions and increases depending on dose levels used and duration of treatment, perhaps compensating for the haemoglobin effect. Haematological effects have been found with a variety of different vanadium compounds including sodium metavanadate, vanadium pentoxide, and ammonium metavanadate supporting the use of this endpoint.

 

The fact that evidence of haematological effects was also observed following 90-day inhalation exposure to vanadium pentoxide, in the absence of other remarkable systemic toxicity (NTP, 2002), increases the confidence in this being the appropriate critical effect for oral exposure from the available dataset. Additional support for the reliability of this endpoint comes from a study by Hogan (2000), where haematological effects were demonstrated following IV injection of three different vanadium compounds each with a different valence state (vanadium chloride (V-III); vanadyl sulphate (V-IV); and sodium orthovanadate (V-V)).

 

Few other signs of systemic toxicity following oral ingestion of vanadium compounds have been reported in other studies, but they do not show a consistent picture. In a study conducted by Domingo et al. (1985), some evidence for renal effects were reported following exposure for 3 months to sodium metavanadate however the effects (increased plasma protein, urea and uric acid) were limited to the top dose with the mid and low dose not being affected. Organ weights, including kidneys, were not affected, and histopathology data were not reported (Domingo et al. (1985). Other studies (Susic and Kentera 1988; NTP 2002) including some assessment of renal function have not shown similar effects although only limited information is available. However, in a study in rats with chronic dietary administration (24 wks) of vanadate (Susic & Kentera, 1988), changes were seen in cardiac output and total peripheral resistance at dose levels of 300 and 3000 ppm NaVO3in the diet. In addition, there was an effect on haematocrit (increase), plasma and blood volume (decrease) as well as extracellular fluid in the high dose group. In another subchronic study (2 months) by Susic & Kentera (1986), 300 ppm represents an effect level for pulmonary function. In this study, no effects were observed on haematocrit levels. The results of a study reported by Jadhav and Jandhyala (1983) suggest that the cardiovascular system responded to vasoconstrictor agents in a dose-dependent manner after subchronic (6 weeks) oral vanadate exposure (drinking water) favouring the development of high blood pressure.In a subchronic study (8 weeks) on behavioural effects of orally administered NaVO3in rats, effects on general activity and learning were observed already at the lowest dose level of 4.1 mg/kg bw/d(Sanchez et al. 1998).Treatment of male rats with different dose levels of vanadyl sulfate in drinking water corresponding to 34, 54 and 90 mg/kg bw/day over 52 weeks did not indicate severe signs of systemic toxicity under the conditions of this study. Body weights were dose-dependently reduced in treatment groups compared to controls, occasionally reaching statistical significance in the low and mid dose groups and at most time points in the high dose group. Based on these effects, the lowest dose level of 34 mg/kg bw/d represents a LOAEL.

 

Based on the available limited data on repeated dose toxicity following oral exposure of soluble tetra- and pentavalent vanadium substances, effects on red blood cell parameters can be regarded as the most robust effect of systemic toxicity. This is further backed by results from a 90-day NTP study (2002) with inhalation exposure of rats. Of the studies showing haematological effects, several of the studies measured the effect with one dose of vanadium compound while other treatment groups received vanadium combined with other substances. These studies are regarded as supportive. Results from the Mountain et al. study (1953) were selected as the starting point for derivation of the DNEL for oral exposure, because it represents the study with the longest duration (103 days) and included several dosage groups. Although the effects observed at the low dose level of 100 ppm V in the diet are only minimal, this dose level is regarded to represent a LOEL in order to protect for potential other toxicological effects. Almost similar results were obtained by Zaporowska et al. (1993) in a 4-week toxicity study, but as the study is only of short-term duration, it is considered as supportive.

Conversion of LOEL (ppm in diet) to LOEL (mg/kg bw /d):

LOEL:             100 ppm V                  Duration:                                 103 d

Food intake:    2,130 g/rat                   Body weight (kg bw/rat):          0.5 kg

V ingested reported:                           155 mg V/rat/103 d= 1.5 mg V/kg

LOELcorrected= 3.0 mg V/kg bw/d based on exposure to soluble vanadium forms

Inhalation:

The most informative study is the standard NTP chronic inhalation study (NTP 2002) using V2O5. In this investigation, there was a statistical increase in lung tumours in mice of both sexes, but not in rats (Starr, 2012). In mice, survival rates of male mice exposed to 4 mg/m3 was less than that of chamber controls, and mean body weights of male mice exposed to 4 mg/m3 and all exposed groups of female mice were generally less than those of the chamber controls throughout the study. As in the 3-month studies, the respiratory tract was the primary site of toxicity. Under the conditions of this 2-year inhalation study there was clear evidence of carcinogenic activity of vanadium pentoxide in male and female B6C3F1 mice based on increased incidences of alveolar/bronchiolar neoplasms. Exposure to vanadium pentoxide caused a spectrum of non-neoplastic lesions in the respiratory tract (nose, larynx, and lung) including alveolar and bronchiolar epithelial hyperplasia, inflammation, fibrosis, and alveolar histiocytosis of the lung in male and female mice. Hyperplasia of the bronchial lymph node occurred in female mice. The lowest concentration tested (1 mg/m3) represents a LOAEC for local effects in the respiratory tract.

Pulmonary reactivity was also investigated in a subchronic inhalation study in cynomolgus monkeys (duration 6 months) with divanadium pentaoxide. The results showed a concentration-dependent impairment in pulmonary function, characterized by airway obstructive changes (pre-exposure challenges) accompanied by a significant influx of inflammatory cells recovered from the lung by bronchoalveolar lavage. Subchronic V2O5 inhalation did not produce an increase in V2O5 reactivity, and cytological, and immunological results indicate the absence of allergic response.

However, local effects on the respiratory tract are not considered relevant for vanadium dioxide for the following reasons:

Severe irritant properties of V2O5have been identified for eye (cat 1) and in lungs, and the redox potential of V2O5 as well as the sharp decline on pH in contact with aqueous media is hypothesised to either mediate this mechanism or at least propagate this mechanism. In contrast, there is no indication whatsoever of any potential for irritation of the respiratory tract for V2O3and VO2. With regard to substance-specific properties assumed to predominantly account for an irritation potential, V2O3 and VO2 are different from V2O5as follows:

- A low dissolution of V2O3was observed in artificial lysosomal fluid (11.9% after 2h; 15.7% after 24h) and lung fluid (4.7% after 2h; 5.6% after 24h)while pentavalent substances (V2O5or NaVO3) dissolved completely within 2 h. Thus, the bioavailability and reactivity of V2O3in tissues of the respiratory tract is assumed to be far less than that of V2O5. Based on similar water solubility and transformation/dissolution characteristics, a similar low bioavailability in the lung is assumed for VO2.

- V2O3and VO2upon contact with water do not cause such significant pH decrease as is the case for V2O5, thus indicating a lack of acidifying properties in aqueous media and any potential for tissue injury associated therewith of V2O3and VO2.

- V2O3and VO2are completely void of oxidising properties and the potential for oxidative injury.

- While VO2lacks any irritation potential to skin and eye in vivo, some very mild but reversible effects have been observed in vivo in the eye after exposure to V2O3.

-V2O3and VO2are not acutely toxic or harmful via inhalation whereas V2O5 is.

Regarding the potential for respiratory irritation, a comprehensive histopathological evaluation of lung tissue was performed within 14-d inhalation studies conducted both with V2O3and V2O5. Severe lung effects including hyperplasia in alveolar and bronchial epithelia, inflammation or fibrosis could not be observed at exposure levels up to 250 mg/m3 with V2O3, whereas these effects are reported as severe for all animals exposed to V2O5already at a level of 2 mg/m3. In conclusion, the onset of marked irritation effects with V2O5 occurs at exposure levels approx. 100-fold lower than with V2O3; on the other hand, given the low solubility and the high exposures, the onset of overload phenomena cannot be completely excluded for V2O3. Based on similar substance-specific properties (as outlined above) this conclusion is read-across to VO2. Thus, it is assumed that vanadium dioxide does not cause respiratory tract irritation.

No carcinogenicity, no pneumoconiosis and no other signs indicative of allergic inflammation have been reported for workers manufacturing vanadium dioxide.Therefore, the local respiratory effects of V2O5 are not relevant for read-across to vanadium dioxide.

The registrant is aware that the National Toxicology Programme (NTP) in the US nominated tetra- and pentavalent vanadium forms(sodium metavanadate, NaVO3, CAS # 13718-26-8; and vanadium oxide sulphate, VOSO4, CAS # 27774-13-6), i.e. species present in drinking water and dietary supplements in 2007 (http://ntp.niehs.nih.gov/). A comprehensive characterisation via the oral route of exposure of

(i) chronic toxicity,

(ii) carcinogenicity, and 

(iii) multi-generation reproductive toxicity

is planned.

 

The NTP testing program began with sub-chronic drinking water and feed studies on VOSO4& NaVO3as follows:

- Genetic toxicology studies, i.e. the Salmonella gene mutation assays, with NaVO3 and VOSO4 - negative

-14 days with Harlan Sprague-Dawley rats and B6C3F1/N mice (dose: R&M: 0, 125, 250, 500, 1000, 2000 mg/L) - already completed

- 90days with Harlan Sprague-Dawley rats and B6C3F1/N mice (dose: R&M:: 0, 31.3, 62.5, 125, 250, or 500 ppm) - ongoing

- Perinatal dose-range finding study: gestation day 6 (GD 6) until postnatal day 42 (PND 42) with Harlan Sprague-Dawley rats - ongoing

- 28days immunotoxicity study (dosed-water) with female B6C3F1/N mice (dose:0, 31.3, 62.5, 125, 250, or 500 ppm) - ongoing

It can reasonably be anticipated that these studies will be of high quality and relevance, and thus will serve as a more robust basis than the current data base with all its shortcomings.In addition, repeated-dose inhalation toxicity studies (14, 28, and 90 days) with various vanadium substances are planned within the Vanadium Safety Readiness Safety Program. These studies will address issues for which to date equivocal or no data at all exist.Further information on these studies can be found in the attachments below.Only upon availability of the results from these studies, it will be possible to render a more meaningful decision on whether or not testing for repeated-dose toxicity is required. Therefore for the time being this data requirement should be waived in consideration of animal welfare.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
A number of studies are available where vanadium compounds were administered; however, they have involved different experimental approaches and designs as well as different dose regimens, and endpoints. For oral exposure, effects are more limited and the different experimental approaches lead to a variety of endpoints measured.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
reliable GLP-conform study with V2O3

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
reliable GLP-conform study with V2O3

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Data of the repeated-dose toxicity via the dermal route are not available for any vanadium substance. Following the HERAG guidance for metals and metal salts (see section 7.1.2 of the technical dossier: dermal absorption), negligible percutaneous uptake based on minimal penetration, i.e. a dermal absorption rate in the range of maximally 0.1 - 1.0 %, can be anticipated. Dermal absorption in this order of magnitude is not considered to be “significant”. Thus, regarding repeated-dose toxicity of vanadium substances, the dermal exposure route is not expected to be the most relevant.

References:
EBRC (2007) HERAG fact sheet - Assessment of occupational dermal exposure and dermal absorption for metals and inorganic metal compounds, EBRC Consulting GmbH, Hannover, Germany, August 2007, 49 pages.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Data of the repeated-dose toxicity via the dermal route are not available for any vanadium substance. Following the HERAG guidance for metals and metal salts (see section 7.1.2 of the technical dossier: dermal absorption), negligible percutaneous uptake based on minimal penetration, i.e. a dermal absorption rate in the range of maximally 0.1 - 1.0 %, can be anticipated. Dermal absorption in this order of magnitude is not considered to be “significant”. Thus, regarding repeated-dose toxicity of vanadium substances, the dermal exposure route is not expected to be the most relevant. In addition, vanadium dioxide does not have any potential for skin irritation as indicated by the lack of any effects in the respective in vivo test.

References:
EBRC (2007) HERAG fact sheet - Assessment of occupational dermal exposure and dermal absorption for metals and inorganic metal compounds, EBRC Consulting GmbH, Hannover, Germany, August 2007, 49 pages.

Justification for classification or non-classification

The currently available and reliable toxicity data on vanadium substances does not justify classification of vanadium dioxide for specific target organ toxicity - repeated exposure.

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