Registration Dossier

Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-01-06 (first exposure) to 1999-01-04/08 (necropsy date)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Well reported study. Test procedure in accordance with generally accepted scientific standards and described in sufficient detail.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990-April-10 (first exposure) to 1990-April-26 (Necropsy)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Well reported study, however study period is shorter than recommended. Test procedure in accordance with generally accepted scientific standards and described in sufficient detail.
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Male and female F344 rats and B6C3F1 mice (N=5/sex/dose, approx. 6 weeks old) were exposed to V2O5 aerosols at concentrations of 0, 2, 4, 8, 16 or 32 mg/m³ by inhalation, 6 hours/d, for 5 d/wk and for a period of 16 days.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Simonsen Laboratories (Gilroy, CA)
- Age at study initiation: average age: 6 weeks old on the first day of the study (4 weeks old on receipt)
- Weight at study initiation: range of mean body weights in the exposure groups: 122-125 g (males) and 101-104 g (females)
- Housing: housed individually; stainless steel wire mesh (Lab Products, Inc., Garfield, NJ), changed weekly
- Diet: ad libitum, except during exposure periods; NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA)
- Water: ad libitum; tap water (City of Chicago municipal supply) via automatic watering system
- Acclimation period: quarantined for 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 23.9± ca.2°C (75° ± 3° F)
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day

IN-LIFE DATES: From: 1990-April-10 (first exposure) To: 1990-April-26 (necropsy date)
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1 - <= 1.3 µm
Remarks on MMAD:
GSD = 2.3-2.8
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- For the 16-day and 3-month studies, vanadium pentoxide aerosol generation was based on the principle of pneumatic dispersion and consisted of two major components: a screw feeder (Model 310, Accurate, White Water, WI) that metered vanadium pentoxide powder at a constant rate and a Jet-O-Mizer jetmill (Fluid Energy Corp., Harfield, PA) that used compressed air to disperse the metered powder and form the aerosol.
- Aerosol leaving the jetmill passed through a one-stage impactor and a vertical elutriator to eliminate or deagglomerate the large particles before entering a plenum and manifold distribution system. The aerosol delivery system consisted of three holding chambers that diluted the aerosol in three stages. A metered amount of diluted aerosol was removed and mixed with conditioned air at the inlet to each exposure chamber to achieve the appropriate exposure concentration. The electrical charge buildup on the aerosol particles was neutralized by mixing the aerosol with high concentrations of bipolar ions, which were generated using a Pulse Gun (Static Control Services, Palm Springs, CA) air nozzle. For the 3-month studies, a transvector air pump was installed at the aerosol inlet to each exposure chamber to provide additional control of the aerosol flow rate and improve stability of the chamber concentration.
- The stainless-steel inhalation exposure chambers (Harford Systems Division of Lab Products, Inc., Aberdeen, MD) were designed so that uniform aerosol concentrations could be maintained throughout the chambers when catch pans were in place. The total active mixing volume of each chamber was 1.7 m³.

CHAMBER ATMOSPHERE CHARACTERIZATION
- The particle size distribution in each chamber was determined prior to the start of all studies, during the first week of the 16-day and 3-month studies, during the first 2 weeks of the 2-year studies, and monthly during the 3-month and 2-year studies.
- For the 16-day and 3-month studies, a 10-stage Quartz Crystal Microbalance-based cascade impactor was used to separate the aerosol particles into sequential size ranges; the mass median aerodynamic diameter was calculated from the corresponding mass fraction of particles at each stage.

OTHER
- The uniformity of aerosol concentration in the inhalation exposure chambers without animals was evaluated before each of the studies began; concentration uniformity with animals present in the chambers was also measured. During the 16-day and 3-month studies, minor excursions in chamber uniformity values were observed in one or more exposure chambers, but these excursions had no impact on the studies. Chamber concentration uniformity was acceptable throughout the 16-day special studies and 2-year studies.
- The stability of vanadium pentoxide in the exposure system was tested with XRD analysis. XRD analyses indicated no detectable build-up of degradation products at a detection limit of approximately 1%.



Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- During all studies, chamber aerosol concentrations were monitored with real-time aerosol monitors (RAMs) that used a pulsed-light-emitting diode in combination with a silicon detector to sense light scattered over a forward angular range of 45° to 95° by particles traversing the sensing volume. The instruments respond to particles 0.1 to 20 μm in diameter.
- During the 16-day and 3-month studies, an individual monitor was used for each exposure chamber. The voltage output of the online monitors was read and recorded, and the calibration curve was applied to the voltages measured by the RAM to convert the measured voltages to exposure chamber concentrations.
- Each RAM was calibrated daily during the 16-day and 3-month studies by correlating the measured voltage with vanadium pentoxide concentrations determined by gravimetric analysis of glass fiber filters and one to two times per week during the 2-year studies by ICP/AES or ICP/mass spectrometry analysis of Pallflex® TX40H120WW glass fiber filters.
Duration of treatment / exposure:
16 days
Frequency of treatment:
6 hours per day, 5 days per week
Dose / conc.:
2 mg/m³ air (nominal)
Dose / conc.:
4 mg/m³ air (nominal)
Dose / conc.:
8 mg/m³ air (nominal)
Dose / conc.:
16 mg/m³ air (nominal)
Dose / conc.:
32 mg/m³ air (nominal)
No. of animals per sex per dose:
Groups of five male and five female rats
Control animals:
yes
Details on study design:
- Dose selection rationale: no data
- Rationale for animal assignment (if not random): randomly into groups of approximately equal initial mean body weights.
Positive control:
not stated
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: observation twice daily; clinical findings were recorded daily.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: core study animals were weighed initially, on day 7, and at the end of the studies; immunotoxicology study animals were weighed initially and at the end of the studies.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

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 data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

IMMUNOLOGY: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
Necropsies were performed on all core study rats.
Method of sacrifice: CO2 asphyxiation. The heart, right kidney, liver, lung, right testis, and thymus of core study animals were weighed.

HISTOPATHOLOGY: Yes.
- Histopathologic examinations were performed on all organs from exposed animals that showed evidence of gross lesions along with corresponding organs of all chamber control animals.
Other examinations:
Special Studies:
- Immunotoxicology studies were performed on day 16 for groups of rats exposed to 0, 4, 8, or 16 mg/m3
- Pulmonary bactericidal activity analyses: lungs from groups of 12 male rats exposed to viable radiolabeled [35S]-K. pneumoniae were evaluated for pulmonary bactericidal activity 3 hours after inoculation.
- Pulmonary lavage analyses: alveolar fluid from 10 male rats was collected by tracheobronchial lavage for measurement of protein concentration and lysozyme activity. Total, viable, and differentiated lavage cell counts were performed. Alveolar macrophages were evaluated for hydrogen peroxide and tumor necrosis factor production and Fc receptormediated phagocytosis (male rats only).
Statistics:
Survival Analyses:
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. Animals found dead of other than natural causes were censored from the survival analyses; animals dying from natural causes were not censored. Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.

Analysis of Continuous Variables:
Two approaches were employed to assess the significance of pairwise comparisons between exposed and control groups in the analysis of continuous variables. Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Hematology, clinical chemistry, urinalysis, urine concentrating ability, cardiopulmonary, immunotoxicologic, cell proliferation, tissue concentrations, spermatid, and epididymal spermatozoal data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test). Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973). Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across exposure concentrations (for more information see publication).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Clinical findings included rapid respiration and hypoactivity in rats exposed to 16 or 32 mg/m³
- From day 8 until the end of the study, rats in the 32 mg/m3 groups became emaciated and had hunched and/or abnormal posture and a rough coat; one of the two surviving males had labored breathing
- Urine staining, salivation, and diarrhea were observed in 32 mg/m³ females
- Ocular or nasal discharge was noted in the 16 mg/m³ groups
Mortality:
mortality observed, treatment-related
Description (incidence):
- 3 males of the 32 mg/m³ group died before the end of the study
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Mean body weights of males and females exposed to 8 mg/m³ or greater were less than those of the chamber controls
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- Relative lung weights of 4 mg/m³ or greater males and 2 mg/m³ or greater females were significantly greater than those of the chamber controls
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Complete histopathology was not performed.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
- A localized inflammatory response in the lung was evident in male rats based on increases in cell number, protein, neutrophils, and lysozymes in lavage fluid in all exposed groups. There was also a significant decrease in macrophages in lavage fluids of male rats exposed to 8 or 16 mg/m3.
Details on results:
ORGAN WEIGHTS
- Further organ weight differences were considered to be related to body weight decreases

GROSS PATHOLOGY
- Gross lesions observed at necropsy were not considered to be exposure related.

HISTOPATHOLOGY: NON-NEOPLASTIC
- Complete histopathology was not performed

OTHER FINDINGS
- There were no effects on systemic immunity, evidenced by a normal response to Klebsiella pneumoniae. Other measures of immune function were
not considered to be significantly different than those of the chamber controls
Dose descriptor:
LOAEC
Remarks:
local
Effect level:
2 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Critical effects observed:
not specified

Based on decreased survival in the 32 mg/m3 males and body weight decreases in 32 mg/m3 males and females, an exposure concentration of 32 mg/m3 was considered too high for use in a 3-month study. Therefore, the exposure concentrations selected for the 3-month inhalation study in rats were 0, 1, 2, 4, 8, and 16 mg/m3.

Conclusions:
Clear signs of toxicity including mortality were observed at the highest concentration level of 32 mg/m3. Clinical signs were observed at 16 and 32 mg/m3, and mean body weights were affected at and above 8 mg/m3. Localised inflammatory responses in lungs were observed in all groups with a decrease in macrophages in lavage fluids of male rats exposed to 8 or 16 mg/m3. Relative lung weights were increased at and above 2 mg/m3. Thus, results of this short-term study suggest a LOAEC of 2 mg/m3 in rats.
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1990-04-11 (first day of exposure) - 1990-04-27 (necropsy date)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well reported study, however study period is shorter than recommended.
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Male and female F344 rats and B6C3F1 mice (N=5/sex/dose, approx. 6 weeks old) were exposed to V2O5 aerosols at concentrations of 0, 2, 4, 8, 16 or 32 mg/m³ by inhalation, 6 hours/d, for 5 d/wk and for a period of 16 days.
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Simonsen Laboratories (Gilroy, CA)
- Age at study initiation: average age: 6 weeks old on the first day of the study (4 weeks old on receipt)
- Weight at study initiation: range of mean body weights in the exposure groups: ca. 23-24 g (males) and ca. 18-19.5 g (females)
- Housing: housed individually; stainless steel wire mesh (Lab Products, Inc., Garfield, NJ), changed weekly
- Diet: ad libitum, except during exposure periods; NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA)
- Water: ad libitum; tap water (City of Chicago municipal supply) via automatic watering system
- Acclimation period: quarantined for 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 23.9± ca.2°C (75° ± 3° F)
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day

IN-LIFE DATES: From: 1990-04-11 (first day of exposure) - 1990-04-27 (necropsy date)
- No further information on test material was stated.
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: MMAD = 1.0-1.3 µm, GSD = 2.3-2.8
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- For the 16-day and 3-month studies, vanadium pentoxide aerosol generation was based on the principle of pneumatic dispersion and consisted of two major components: a screw feeder (Model 310, Accurate, White Water, WI) that metered vanadium pentoxide powder at a constant rate and a Jet-O-Mizer jetmill (Fluid Energy Corp., Harfield, PA) that used compressed air to disperse the metered powder and form the aerosol.
- Aerosol leaving the jetmill passed through a one-stage impactor and a vertical elutriator to eliminate or deagglomerate the large particles before entering a plenum and manifold distribution system. The aerosol delivery system consisted of three holding chambers that diluted the aerosol in three stages. A metered amount of diluted aerosol was removed and mixed with conditioned air at the inlet to each exposure chamber to achieve the appropriate exposure concentration. The electrical charge buildup on the aerosol particles was neutralized by mixing the aerosol with high concentrations of bipolar ions, which were generated using a Pulse Gun (Static Control Services, Palm Springs, CA) air nozzle. For the 3-month studies, a transvector air pump was installed at the aerosol inlet to each exposure chamber to provide additional control of the aerosol flow rate and improve stability of the chamber concentration.
- The stainless-steel inhalation exposure chambers (Harford Systems Division of Lab Products, Inc., Aberdeen, MD) were designed so that uniform aerosol concentrations could be maintained throughout the chambers when catch pans were in place. The total active mixing volume of each chamber was 1.7 m³.

CHAMBER ATMOSPHERE CHARACTERIZATION
- The particle size distribution in each chamber was determined prior to the start of all studies, during the first week of the 16-day and 3-month studies, during the first 2 weeks of the 2-year studies, and monthly during the 3-month and 2-year studies.
- For the 16-day and 3-month studies, a 10-stage Quartz Crystal Microbalance-based cascade impactor was used to separate the aerosol particles into sequential size ranges; the mass median aerodynamic diameter was calculated from the corresponding mass fraction of particles at each stage.

OTHER
- The uniformity of aerosol concentration in the inhalation exposure chambers without animals was evaluated before each of the studies began; concentration uniformity with animals present in the chambers was also measured. During the 16-day and 3-month studies, minor excursions in chamber uniformity values were observed in one or more exposure chambers, but these excursions had no impact on the studies. Chamber concentration uniformity was acceptable throughout the 16-day special studies and 2-year studies.
- The stability of vanadium pentoxide in the exposure system was tested with XRD analysis. XRD analyses indicated no detectable build-up of degradation products at a detection limit of approximately 1%.



Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- During all studies, chamber aerosol concentrations were monitored with real-time aerosol monitors (RAMs) that used a pulsed-light-emitting diode in combination with a silicon detector to sense light scattered over a forward angular range of 45° to 95° by particles traversing the sensing volume. The instruments respond to particles 0.1 to 20 μm in diameter.
- During the 16-day and 3-month studies, an individual monitor was used for each exposure chamber. The voltage output of the online monitors was read and recorded, and the calibration curve was applied to the voltages measured by the RAM to convert the measured voltages to exposure chamber concentrations.
- Each RAM was calibrated daily during the 16-day and 3-month studies by correlating the measured voltage with vanadium pentoxide concentrations determined by gravimetric analysis of glass fiber filters and one to two times per week during the 2-year studies by ICP/AES or ICP/mass spectrometry analysis of Pallflex® TX40H120WW glass fiber filters.
Duration of treatment / exposure:
16 days
Frequency of treatment:
6 hours per day, 5 days per week
Remarks:
Doses / Concentrations:
2 mg/m³ V2O5
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
4 mg/m³ V2O5
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
8 mg/m³ V2O5
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
16 mg/m³ V2O5
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
32 mg/m³ V2O5
Basis:
nominal conc.
No. of animals per sex per dose:
Groups of five male and five female mice were exposed.
Control animals:
yes
Details on study design:
- Dose selection rationale: no data
- Rationale for animal assignment (if not random): randomly into groups of approximately equal initial mean body weights.
- No further information on test material was stated.
Positive control:
no data
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily; clinical findings were recorded daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: core study animals were weighed initially, on day 7, and at the end of the studies; immunotoxicology study animals were weighed initially and at the end of the studies

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

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 data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Necropsies were performed on all core study mice.
Method of sacrifice: CO2 asphyxiation. The heart, right kidney, liver, lung, right testis, and thymus of core study animals were weighed.

HISTOPATHOLOGY: Yes.
- Histopathologic examinations were performed on all organs from exposed animals that showed evidence of gross lesions along with corresponding organs of all chamber control animals.
Other examinations:
- Groups of five male and five female rats and mice were exposed to particulate aerosols of vanadium pentoxide at concentrations of 0, 2, 4, 8, 16, or 32 mg/m3, 6 hours plus T90 (15 minutes) per day, 5 days per week for 16 days
- Additional groups of 50 female mice designated for immunotoxicology studies were exposed to 0, 4, 8, or 16 mg/m3 for 16 days
- Influenza virus challenge: groups of 20 female mice were instilled intranasally with influenza virus and evaluated for moribundity for 14 days.
- Pulmonary bactericidal activity analyses: lungs from groups of 12 female mice exposed to viable radiolabeled [35S]-K. pneumoniae were evaluated for pulmonary bactericidal activity 3 hours after inoculation.
- Pulmonary lavage analyses: alveolar fluid from 10 female mice was collected by tracheobronchial lavage for measurement of protein concentration and lysozyme activity. Total, viable, and differentiated lavage cell counts were performed.
- Mixed lymphocyte culture response/cytotoxic T cell response analyses: groups of eight female mice were evaluated for mixed lymphocyte response to allogenic splenocytes and induction of cytotoxic T lymphocytes.
Statistics:
Survival Analyses:
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. Animals found dead of other than natural causes were censored from the survival analyses; animals dying from natural causes were not censored. Statistical analyses for possible doserelated effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.

Analysis of Continuous Variables:
Two approaches were employed to assess the significance of pairwise comparisons between exposed and control groups in the analysis of continuous variables. Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Hematology, clinical chemistry, urinalysis, urine concentrating ability, cardiopulmonary, immunotoxicologic, cell proliferation, tissue concentrations, spermatid, and epididymal spermatozoal data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test). Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973). Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across exposure concentrations. (for more information see publication)
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
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:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
complete histopathology was not done, grossly enlarged nodes were confirmed histologically as lymphoid hyperplasia
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
- All males exposed to 32 mg/m3 and one 8 mg/m3 male died or were killed moribund before the end of the study
- Hypoactivity was observed in the 32 mg/m3 groups; one of the affected females also had labored breathing
- Some males in the 32 mg/m3 groups had hunched posture, and one was emaciated

BODY WEIGHT AND WEIGHT GAIN
- Body weight deficits greater than 10% were observed in 32 mg/m3 mice
- Final mean body weights and body weight gains of 16 mg/m3 males and 32 mg/m3 females were significantly less than those of the chamber controls; and the 32 mg/m3 females lost weight during the study
- Additionally, final mean body weights of 8 and 16 mg/m3 females were significantly less than those of the chamber controls

ORGAN WEIGHTS
- Absolute and relative lung weights of 4 mg/m3 or greater males and all exposed groups of females and liver weights of 16 mg/m3 males were significantly greater than those of the chamber controls
- Liver weights of 16 mg/m3 males were significantly greater than those of the chamber controls
- Other organ weight differences were considered to be related to body weight decreases
- Thymus weights were similar to those of the chamber controls in all exposed groups except 32 mg/m3 females

HISTOPATHOLOGY: NON-NEOPLASTIC
- While complete histopathology was not done, grossly enlarged lymph nodes were confirmed histologically as lymphoid hyperplasia (data not presented)

OTHER FINDINGS
- The mediastinal lymph nodes were enlarged in 4, 8, and 16 mg/m3 males and females, and lymphoid hyperplasia was confirmed histologically
- A localized inflammatory response in the lung was evident based on increases in cell number, protein, lymphocytes, neutrophils, and lysozymes in lavage fluid. There was also a significant decrease in macrophages in lavage fluid.
- There were no effects on systemic immunity as evidenced by normal responses to Klebsiella pneumoniae and influenza virus
- Other measures of immune function were not considered to be significantly different than those of the chamber controls
Dose descriptor:
LOAEC
Remarks:
local
Effect level:
2 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: increased absolute and relative lung weights and inflammatory responses in the lungs
Critical effects observed:
not specified

Based on decreased survival of 32 mg/m3 males and body weight reduction in the 32 mg/m3 females, exposure concentrations greater than 16 mg/m3 were considered too high for use in a 3-month study. Therefore, the exposure concentrations selected for the 3-month inhalation study in mice were 0, 1, 2, 4, 8, and 16 mg/m3.

Conclusions:
Clinical signs of toxicity and mortality were observed at the highest concentration of 32 mg/m3. Body weights were affected at and above 8 mg/m3 and absolute and relative lung weights at and above 2 mg/m3. A localized inflammatory response in the lung was evident. Therefore, results of this short-term study suggest a LOAEC of 2 mg/m3 in mice.
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1996-02-08 (first exposure) to 1996- 02 -23 (necropsy date; max.)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well reported study, however study period is shorter than recommended. Test procedure in accordance with generally accepted scientific standards and described in sufficient detail.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Beside the core study, tissue burden studies were conducted in female rats to determine lung and blood vanadium concentrations and lung clearance half-times of vanadium in anticipation of measuring tissue burdens during the 2-year studies. Groups of 60 female rats were exposed to particulate aerosols of vanadium pentoxide at concentrations of 0, 1, or 2 mg/m3 and a group of 40 female rats was exposed to 4 mg/m3 by inhalation, 6 hours per day, 5 days per week for 16 days.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: average age: 6 weeks old on the first day of the study (4 weeks old on receipt)
- Housing: housed individually; stainless steel wire bottom (Hazleton Systems, Inc., Aberdeen, MD), changed weekly
- Diet: ad libitum, except during exposure periods; NTP-2000 pelleted diet (Zeigler Brothers, Inc., Gardeners, PA)
- Water: ad libitum; tap water (Richland, WA, municipal supply water) via automatic watering system
- Acclimation period: quarantined for 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 23.9± ca.2°C (75° ± 3° F)
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day

IN-LIFE DATES: From: 1996-02-08 (first exposure) to 1996- 02 -23 (necropsy date; max.)
- No further information on test material was stated.
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: MMAD = 1.0-1.3 µm, GSD = 2.3-2.8
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- The generation and delivery system used in the 16-day special studies and the 2-year studies consisted of a linear dust feeder, a particle attrition chamber, and an aerosol distribution system. The linear dust feeder, a slide-bar dust-metering device, was composed of a shuttle bar, body, outlet port, and hopper. As the compressed-air-driven shuttle bar slid back and forth during generation, the metering port aligned with the hopper, which served as a reservoir for the bulk chemical, and was filled with a small amount of vanadium pentoxide powder. As the shuttle bar slid to the dispersing position, the metering port aligned with a compressed-air port in the body and a puff of air from this port dispersed the vanadium pentoxide into the particle attrition chamber. Generator output was regulated by adjusting the cadence of the shuttle bar. The particle attrition chamber used low fluid energy from an air jet tangential to the chamber to deagglomerate the vanadium pentoxide particles. After deagglomeration, the particles were swept into a classification zone where smaller particles exited to the distribution line; larger particles were thrown to the perimeter of the classifier by centrifugal force and were reentrained into the impacting air jet, and the process was repeated until the particles were sufficiently deagglomerated. The aerosol passed through the distribution lines to the exposure chambers. A pneumatic pump designed by the study laboratory was located at each chamber inlet and drew aerosol from the distribution line into the chamber inlet, where it was diluted with conditioned air to the appropriate concentration. Flow through the distribution line was controlled by Air-Vac pumps, and pressure was monitored by photohelic differential pressure gauges.
- The stainless-steel inhalation exposure chambers (Lab Products, Inc., Harford Systems Division, Aberdeen, MD), were designed so that uniform aerosol concentrations could be maintained throughout the chambers when catch pans were in place. The total active mixing volume of each chamber was 1.7 m³.

CHAMBER ATMOSPHERE CHARACTERIZATION
- The particle size distribution in each chamber was determined prior to the start of all studies, during the first week of the 16-day and 3-month studies, during the first 2 weeks of the 2-year studies, and monthly during the 3-month and 2-year studies.
- For the 16-day special studies and the 2-year studies, a Mercer-style seven-stage impactor was used. The stages (glass coverslips lightly sprayed with silicon) were analyzed by ICP/AES, and the relative mass collected on each stage was analyzed by probit analysis

OTHER
- The uniformity of aerosol concentration in the inhalation exposure chambers without animals was evaluated before each of the studies began; concentration uniformity with animals present in the chambers was also measured. During the 16-day and 3-month studies, minor excursions in chamber uniformity values were observed in one or more exposure chambers, but these excursions had no impact on the studies. Chamber concentration uniformity was acceptable throughout the 16-day special studies and 2-year studies.
- The stability of vanadium pentoxide in the exposure system was tested with XRD analysis. XRD analyses indicated no detectable build-up of degradation products at a detection limit of approximately 1%.



Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- During all studies, chamber aerosol concentrations were monitored with real-time aerosol monitors (RAMs) that used a pulsed-light-emitting diode in combination with a silicon detector to sense light scattered over a forward angular range of 45° to 95° by particles traversing the sensing volume. The instruments respond to particles 0.1 to 20 μm in diameter.
- For the 16-day special studies and the 2-year studies, the sampling system consisted of a valve that multiplexed each RAM to two or three exposure chambers and to a HEPA filter and/or the control chamber or room; selection of sampling streams and data acquisition from each RAM was remotely controlled by a computer. Equations for calibration curves were stored in the computers and were used to convert the measured voltages to exposure concentrations.
- Each RAM was calibrated daily during the 16-day and 3-month studies by correlating the measured voltage with vanadium pentoxide concentrations determined by gravimetric analysis of glass fiber filters and one to two times per week during the 2-year studies by ICP/AES or ICP/mass spectrometry analysis of Pallflex® TX40H120WW glass fiber filters.
Duration of treatment / exposure:
16 days
Frequency of treatment:
6 hours per day, 5 days per week
Remarks:
Doses / Concentrations:
1 mg/m³ V2O5 (60 female rats)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2 mg/m³ V2O5 (60 female rats)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
4 mg/m³ V2O5 (40 female rats)
Basis:
nominal conc.
No. of animals per sex per dose:
Three groups of 60 female rats: control, 1 mg/m³ and 2 mg/m³
One group of 40 females: 4 mg/m³
Control animals:
yes
Details on study design:
- Dose selection rationale: no data
- Rationale for animal assignment (if not random): randomly into groups of approximately equal initial mean body weights.
- No further information on test material was stated.
Positive control:
not stated
Observations and examinations performed and frequency:
OTHER:
- Cell proliferation studies: on days 6 and 13, the lungs of 10 rats per group were evaluated for cell proliferation by measuring the incorporation of BrdU implanted 140 ± 3 hours earlier and were reviewed histopathologically.
- Tissue burden studies: lung and blood from groups of five animals exposed to 0, 1 (rats only), 2, or 4 (mice only) mg/m3 were evaluated on postexposure days 0, 1, 4, and 8. Lung weight, lung burden, and blood vanadium concentration were measured. Female rats were exposed to 0, 1, or 2 mg/m3 vanadium pentoxide for 14 days. Elimination of vanadium from these tissues for a period up to 8 days after exposure was also determined.
- Histopathology: the onset and extent of lung lesions in 0, 1, 2, or 4 mg/m3 females were evaluated in lung tissue collected from four rats per group on days 1, 2, 5, 10, and 16.
Sacrifice and pathology:
Method of Sacrifice: CO2 asphyxiation. Necropsy was performed on four animals per group on days 1, 2, 5, 10, and 16.

GROSS PATHOLOGY: No

HISTOPATHOLOGY: Yes.
- Because histopathology of the lung was not performed in the 16-day studies, histopathology and cell proliferation analyses of the lung following short-term exposure were performed to assess the early lung effects with the intent to possibly conduct mechanistic studies independently or during the 2-year studies. Histopathology was performed on lung tissue from four animals in each exposure group on days 1, 2, 5, 10, and 16.
Statistics:
Survival Analyses: The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. Animals found dead of other than natural causes were censored from the survival analyses; animals dying from natural causes were not censored. Statistical analyses for possible doserelated effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.

Analysis of Continuous Variables:
Two approaches were employed to assess the significance of pairwise comparisons between exposed and control groups in the analysis of continuous variables. Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Hematology, clinical chemistry, urinalysis, urine concentrating ability, cardiopulmonary, immunotoxicologic, cell proliferation, tissue concentrations, spermatid, and epididymal spermatozoal data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test). Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973). Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across exposure concentrations. (for more information see publication)
Clinical signs:
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
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
Description (incidence and severity):
lungs
Gross pathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
ORGAN WEIGHTS
- Lung weights of exposed rats were significantly greater than those of the chamber controls; there was little difference in lung weights among exposed groups
- The lung weights of exposed rats remained greater than those of the chamber controls through day 4 of recovery but were similar to those of the chamber controls on day 8 the exposed groups

OTHER FINDINGS
- Alveolar and bronchiolar epithelial hyperplasia was observed in most rats exposed to 2 or 4 mg/m3 on days 6 and 13
- Histiocytic infiltration and inflammation occurred in a time- and concentration-related manner
- Cell turnover rates were increased in the terminal bronchioles on days 6 and 13 and in the alveoli in the 4 mg/m3 group on day 6 and in all exposed groups on day 13

LUNG
- Lung burdens were proportional to exposure concentration throughout the recovery period; the areas under the lung burden curves suggest proportionality between exposure concentration and lung burden
- Assessment of lung vanadium concentrations suggested deposition and clearance exhibited linear kinetics over the exposure range studied
- Lung clearance half-times ranged from 4.42 to 4.96 days; calculated lung clearance half-times during the 8-day recovery period were not significantly different among exposure groups

BLOOD
- Vanadium was detected in blood at concentrations several orders of magnitude less than those observed in lung tissue from exposed rats
- Blood vanadium concentrations in exposed and chamber control rats were for the most part highly variable and occurred at concentrations slightly above the limit of quantitation
- The marginal increases in blood vanadium concentrations in exposed rats indicate that either very little vanadium was absorbed or it was eliminated rapidly from the blood
Critical effects observed:
not specified
Conclusions:
The 16-day special study was conducted to determine blood and lung concentrations of vanadium, the lung clearance half-time of vanadium, and the onset and extent of vanadium pentoxide-induced lung injury. Lung weights of all exposed groups of rats were significantly greater than those of the chamber controls; there was little difference among exposed groups. After 8 days of recovery, lung weights of exposed rats were similar to those of the chamber controls. Lung burdens and AUCs indicated lung burden proportionality and, when normalized to exposure concentration, indicated linear kinetics.
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1996-02-08 (first exposure) to 1996- 02 -23 (necropsy date; max.)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well reported study, however study period is shorter than recommended. Test procedure in accordance with generally accepted scientific standards and described in sufficient detail.
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Beside the core study, tissue burden studies were conducted in female mice to determine lung and blood vanadium concentrations and lung clearance half-times of vanadium in anticipation of measuring tissue burdens during the 2-year studies. Groups of 60 female mice were exposed to particulate aerosols of vanadium pentoxide at concentrations of 0, 2, or 4 mg/m3 and a group of 40 female mice was exposed to 8 mg/m3 by inhalation, 6 hours per day, 5 days per week for 16 days.
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: average age: 6 weeks old on the first day of the study (4 weeks old on receipt)
- Housing: housed individually; stainless steel wire bottom (Hazleton Systems, Inc., Aberdeen, MD), changed weekly
- Diet: ad libitum, except during exposure periods; NTP-2000 pelleted diet (Zeigler Brothers, Inc., Gardeners, PA)
- Water: ad libitum; tap water (Richland, WA, municipal supply water) via automatic watering system
- Acclimation period: quarantined for 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 23.9± ca.2°C (75° ± 3° F)
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day

IN-LIFE DATES: From: 1996-02-08 (first exposure) to 1996- 02 -23 (necropsy date; max.)
- No further information on test material was stated.
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: MMAD = 1.0-1.3 µm, GSD = 2.3-2.8
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- The generation and delivery system used in the 16-day special studies and the 2-year studies consisted of a linear dust feeder, a particle attrition chamber, and an aerosol distribution system. The linear dust feeder, a slide-bar dust-metering device, was composed of a shuttle bar, body, outlet port, and hopper. As the compressed-air-driven shuttle bar slid back and forth during generation, the metering port aligned with the hopper, which served as a reservoir for the bulk chemical, and was filled with a small amount of vanadium pentoxide powder. As the shuttle bar slid to the dispersing position, the metering port aligned with a compressed-air port in the body and a puff of air from this port dispersed the vanadium pentoxide into the particle attrition chamber. Generator output was regulated by adjusting the cadence of the shuttle bar. The particle attrition chamber used low fluid energy from an air jet tangential to the chamber to deagglomerate the vanadium pentoxide particles. After deagglomeration, the particles were swept into a classification zone where smaller particles exited to the distribution line; larger particles were thrown to the perimeter of the classifier by centrifugal force and were reentrained into the impacting air jet, and the process was repeated until the particles were sufficiently deagglomerated. The aerosol passed through the distribution lines to the exposure chambers. A pneumatic pump designed by the study laboratory was located at each chamber inlet and drew aerosol from the distribution line into the chamber inlet, where it was diluted with conditioned air to the appropriate concentration. Flow through the distribution line was controlled by Air-Vac pumps, and pressure was monitored by photohelic differential pressure gauges.
- The stainless-steel inhalation exposure chambers (Lab Products, Inc., Harford Systems Division, Aberdeen, MD), were designed so that uniform aerosol concentrations could be maintained throughout the chambers when catch pans were in place. The total active mixing volume of each chamber was 1.7 m³.

CHAMBER ATMOSPHERE CHARACTERIZATION
- The particle size distribution in each chamber was determined prior to the start of all studies, during the first week of the 16-day and 3-month studies, during the first 2 weeks of the 2-year studies, and monthly during the 3-month and 2-year studies.
- For the 16-day special studies and the 2-year studies, a Mercer-style seven-stage impactor was used. The stages (glass coverslips lightly sprayed with silicon) were analyzed by ICP/AES, and the relative mass collected on each stage was analyzed by probit analysis

OTHER
- The uniformity of aerosol concentration in the inhalation exposure chambers without animals was evaluated before each of the studies began; concentration uniformity with animals present in the chambers was also measured. During the 16-day and 3-month studies, minor excursions in chamber uniformity values were observed in one or more exposure chambers, but these excursions had no impact on the studies. Chamber concentration uniformity was acceptable throughout the 16-day special studies and 2-year studies.
- The stability of vanadium pentoxide in the exposure system was tested with XRD analysis. XRD analyses indicated no detectable build-up of degradation products at a detection limit of approximately 1%.



Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- During all studies, chamber aerosol concentrations were monitored with real-time aerosol monitors (RAMs) that used a pulsed-light-emitting diode in combination with a silicon detector to sense light scattered over a forward angular range of 45° to 95° by particles traversing the sensing volume. The instruments respond to particles 0.1 to 20 μm in diameter.
- For the 16-day special studies and the 2-year studies, the sampling system consisted of a valve that multiplexed each RAM to two or three exposure chambers and to a HEPA filter and/or the control chamber or room; selection of sampling streams and data acquisition from each RAM was remotely controlled by a computer. Equations for calibration curves were stored in the computers and were used to convert the measured voltages to exposure concentrations.
- Each RAM was calibrated daily during the 16-day and 3-month studies by correlating the measured voltage with vanadium pentoxide concentrations determined by gravimetric analysis of glass fiber filters and one to two times per week during the 2-year studies by ICP/AES or ICP/mass spectrometry analysis of Pallflex® TX40H120WW glass fiber filters.
Duration of treatment / exposure:
16 days
Frequency of treatment:
6 hours per day, 5 days per week
Remarks:
Doses / Concentrations:
2 mg/m³ V2O5 (60 female mice)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
4 mg/m³ V2O5 (60 female mice)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
8 mg/m³ V2O5 (40 female mice)
Basis:
nominal conc.
No. of animals per sex per dose:
Three groups of 60 female mice: control, 2 mg/m³ and 4 mg/m³
One group of 40 females: 8 mg/m³ group
Control animals:
yes
Details on study design:
- Dose selection rationale: no data
- Rationale for animal assignment (if not random): randomly into groups of approximately equal initial mean body weights.
- No further information on test material was stated.
Positive control:
not stated
Observations and examinations performed and frequency:
OTHER:
- Tissue burden studies: lung and blood from groups of five animals exposed to 0, 1 (rats only), 2, or 4 (mice only) mg/m3 were evaluated on postexposure days 0, 1, 4, and 8. Lung weight, lung burden, and blood vanadium concentration were measured.
- Elimination of vanadium from these tissues for a period up to 8 days postexposure was also determined.
- Cell proliferation rates were determined from lung tissue taken from 10 animals per group on days 6 and 13;
- Microscopic examination of lung tissue was conducted on an additional 10 animals per group at each of these time points.
- In addition, the onset and extent of lung lesions in 0, 2, 4, or 8 mg/m3 females were evaluated in lung tissue collected from four animals per group on days 1, 2, 5, 10, and 16
Sacrifice and pathology:
Method of Sacrifice: CO2 asphyxiation. Necropsy was performed on four animals per group on days 1, 2, 5, 10, and 16.

GROSS PATHOLOGY: No

HISTOPATHOLOGY: Yes.
- Because histopathology of the lung was not performed in the 16-day studies, histopathology and cell proliferation analyses of the lung following short-term exposure were performed to assess the early lung effects with the intent to possibly conduct mechanistic studies independently or during the 2-year studies. Histopathology was performed on lung tissue from four animals in each exposure group on days 1, 2, 5, 10, and 16.
Statistics:
Analysis of Continuous Variables:
Two approaches were employed to assess the significance of pairwise comparisons between exposed and control groups in the analysis of continuous variables. Cell proliferation and tissue concentrations, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test). Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973). Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across exposure concentrations. (for more information see publication)
Clinical signs:
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
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
Description (incidence and severity):
lungs
Gross pathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
ORGAN WEIGHTS
- Lung weights of exposed mice were significantly greater than those of the chamber controls; there was little difference in lung weights among the exposed groups
- The lung weights of exposed mice remained greater than those of the chamber controls through day 4 of recovery but were similar to those of the chamber controls on day 8
- In general, lung burdens were proportional to exposure concentration throughout the recovery period

HISTOPATHOLOGY: NON-NEOPLASTIC
- The lungs were evaluated histologically from four mice exposed for 1, 2, 5, 10, or 16 days (data not shown)
- Lungs from animals exposed for 1 or 2 days revealed no significant differences between chamber control and exposed groups
- Lesions similar to those described above were observed on days 5, 10, and 16 with interstitial inflammation first observed at 10 days
- As above, lesions were minimal to mild and tended to increase in incidence and/or severity with time and increasing exposure concentration

OTHER FINDINGS
LUNG
- Alveolar and bronchiolar epithelial hyperplasia occurred with similar incidences and severities among the exposed groups on days 6 and 13, and time- and concentration-related increases in the incidences of interstitial inflammation and histiocytic infiltration also occurred in these groups
- Cell turnover rates were increased in the terminal bronchioles on day 6 and remained greater than those of the chamber controls on day 13
- In the alveoli, cell turnover rates were increased in an exposure concentration-related manner on day 13; cell turnover rates were increased only in the 8 mg/m3 group on day 6
- In general, lung burdens were proportional to exposure concentration throughout the recovery period
- Assessment of lung vanadium concentrations suggested deposition and clearance exhibited linear kinetics over the exposure range studied
- Lung clearance half-times ranged from 2.40 to 2.55 days (mice did clear vanadium from their lungs much faster than did rats)

BLOOD
- Although vanadium was detected in the blood of exposed and chamber control mice, blood vanadium concentrations were highly variable
- In most instances, blood vanadium concentrations in chamber control as well as in exposed mice were below the limit of quantitation
Critical effects observed:
not specified
Conclusions:
The 16-day special study was conducted to determine blood and lung concentrations of vanadium, the lung clearance half-time of vanadium, and the onset and extent of vanadium pentoxide-induced lung injury.Following exposure to vanadium pentoxide, lung weights of all exposed groups of mice were significantly greater than those of the chamber controls; there was little difference among exposed groups. After 8 days of recovery, lung weights of exposed mice were similar to those of the chamber controls. Lung burdens and AUCs indicated lung burden proportionality and, when normalized to exposure concentration, indicated linear kinetics. Mice cleared vanadium from their lungs faster than rats based on lung clearance half-times.
Reason / purpose:
reference to same study
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990-09-18/19 (first exposure) to 1990-12-18/19 (necropsy date)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Well reported study. Test procedure in accordance with generally accepted scientific standards and described in sufficient detail.
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 10 male and 10 female rats were exposed to particulate aerosols of vanadium pentoxide at concentrations of 0, 1, 2, 4, 8, or 16 mg/m3 by inhalation, 6 hours per day, 5 days per week for 3 months. The 3-month studies were conducted to evaluate the cumulative toxic effects of repeated exposure to vanadium pentoxide and to determine the appropriate exposure concentrations to be used in the 2-year studies.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: average age: 6 or 7 weeks old on the first day of the study (4 weeks old on receipt)
- Weight at study initiation: range of mean body weights in the exposure groups: 133-137 g (males) and 108-111 g (females)
- Housing: housed individually; stainless steel wire mesh (Lab Products, Inc., Maywood, NJ),), changed weekly
- Diet: ad libitum, except during exposure periods; NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA); feed was also
withheld during urine collection
- Water: ad libitum; tap water (City of Chicago municipal supply) via automatic watering system; water was withheld for the second urine collection
- Acclimation period: quarantined for 12 or 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 23.9± ca.2°C (75° ± 3° F)
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1 - <= 1.3 µm
Remarks on MMAD:
GSD = 2.3-2.8
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- For the 16-day and 3-month studies, vanadium pentoxide aerosol generation was based on the principle of pneumatic dispersion and consisted of two major components: a screw feeder (Model 310, Accurate, White Water, WI) that metered vanadium pentoxide powder at a constant rate and a Jet-O-Mizer jetmill (Fluid Energy Corp., Harfield, PA) that used compressed air to disperse the metered powder and form the aerosol.
- Aerosol leaving the jetmill passed through a one-stage impactor and a vertical elutriator to eliminate or deagglomerate the large particles before entering a plenum and manifold distribution system. The aerosol delivery system consisted of three holding chambers that diluted the aerosol in three stages. A metered amount of diluted aerosol was removed and mixed with conditioned air at the inlet to each exposure chamber to achieve the appropriate exposure concentration. The electrical charge buildup on the aerosol particles was neutralized by mixing the aerosol with high concentrations of bipolar ions, which were generated using a Pulse Gun (Static Control Services, Palm Springs, CA) air nozzle. For the 3-month studies, a transvector air pump was installed at the aerosol inlet to each exposure chamber to provide additional control of the aerosol flow rate and improve stability of the chamber concentration.
- The stainless-steel inhalation exposure chambers (Lab Products, Inc., Maywood NJ), were designed so that uniform aerosol concentrations could be maintained throughout the chambers when catch pans were in place. The total active mixing volume of each chamber was 1.7 m³.

CHAMBER ATMOSPHERE CHARACTERIZATION
- The particle size distribution in each chamber was determined prior to the start of all studies, during the first week of the 16-day and 3-month studies, during the first 2 weeks of the 2-year studies, and monthly during the 3-month and 2-year studies.
- For the 16-day and 3-month studies, a 10-stage Quartz Crystal Microbalance-based cascade impactor was used to separate the aerosol particles into sequential size ranges; the mass median aerodynamic diameter was calculated from the corresponding mass fraction of particles at each stage.

OTHER
- The uniformity of aerosol concentration in the inhalation exposure chambers without animals was evaluated before each of the studies began; concentration uniformity with animals present in the chambers was also measured. During the 16-day and 3-month studies, minor excursions in chamber uniformity values were observed in one or more exposure chambers, but these excursions had no impact on the studies. Chamber concentration uniformity was acceptable throughout the 16-day special studies and 2-year studies.
- The stability of vanadium pentoxide in the exposure system was tested with XRD analysis. XRD analyses indicated no detectable build-up of degradation products at a detection limit of approximately 1%.



Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- During all studies, chamber aerosol concentrations were monitored with real-time aerosol monitors (RAMs) that used a pulsed-light-emitting diode in combination with a silicon detector to sense light scattered over a forward angular range of 45° to 95° by particles traversing the sensing volume. The instruments respond to particles 0.1 to 20 μm in diameter.
- During the 16-day and 3-month studies, an individual monitor was used for each exposure chamber. The voltage output of the online monitors was read and recorded, and the calibration curve was applied to the voltages measured by the RAM to convert the measured voltages to exposure chamber concentrations.
- Each RAM was calibrated daily during the 16-day and 3-month studies by correlating the measured voltage with vanadium pentoxide concentrations determined by gravimetric analysis of glass fiber filters and one to two times per week during the 2-year studies by ICP/AES or ICP/mass spectrometry analysis of Pallflex® TX40H120WW glass fiber filters.
Duration of treatment / exposure:
3 months (91-92 days)
Frequency of treatment:
6 hours per day, 5 days per week
Dose / conc.:
1 mg/m³ air (nominal)
Dose / conc.:
2 mg/m³ air (nominal)
Dose / conc.:
4 mg/m³ air (nominal)
Dose / conc.:
8 mg/m³ air (nominal)
Dose / conc.:
16 mg/m³ air (nominal)
No. of animals per sex per dose:
- core study: groups of 10 male and 10 female rats
- clinical pathology and cardiopulmonary physiology analyses:groups of 10 male and 10 female rats
Control animals:
yes
Details on study design:
- Dose selection rationale: based on decreased survival in the 32 mg/m3 males and body weight decreases in 32 mg/m3 males and females, an exposure concentration of 32 mg/m3 was considered too high for use in a 3-month study. Therefore, the exposure concentrations selected for the 3-month inhalation study in rats were 0, 1, 2, 4, 8, and 16 mg/m3.
- Rationale for animal assignment (if not random): randomly into groups of approximately equal initial mean body weights.
Positive control:
not stated
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were observed twice daily; clinical findings were recorded weekly

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: the body weight was controlled initially, weekly and at study end

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

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 data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: from the cardiopulmonary physiology study rats on days 4 and 23 and from core study rats at the end of
the studies
- Anaesthetic used for blood collection: rats were anesthetized with a 70% CO2:30% air mixture
- Animals fasted: no data
- How many animals: mainly 8-10 per exposure group (except week 13, highest dose group: N=3 for males and 6 for females)
- Parameters checked: automated hematocrit; manual hematocrit; hemoglobin concentration; erythrocyte, reticulocyte, nucleated erythrocyte, and platelet counts; erythrocyte morphology; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; leukocyte count and differentials

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: from the cardiopulmonary physiology study rats on days 4 and 23 and from core study rats at the end of
the studies
- Animals fasted: no data
- How many animals: mainly 8-10 per exposure group (except week 13, highest dose group: N=3 for males and 6 for females)
- Parameters checked: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, bile acids

URINALYSIS: Yes
- Time schedule for collection of urine: during week 12 (females) or 13 (males) (core study animals)
- Metabolism cages used for collection of urine: for 16 hours immediately following exposure
- Animals fasted: yes
- Parameters checked: urinalysis: volume, specific gravity; urine concentrating ability: volume, specific gravity

IMMUNOLOGY: Not specified

NEUROBEHAVIOURAL EXAMINATION: No data

OTHER:
- Cardiopulmonary physiology studies: cardiopulmonary physiology studies were performed during week 12 (female) or 13 (male) for groups of 10 male and 10 female rats exposed to 0, 4, 8, or 16 g/m³.
- Cardiovascular analyses: heart rate; systolic, diastolic, and mean blood pressure; PR, QRS, and QT intervals; axis shift
- Pulmonary function analyses:
- Tidal breathing measurements: respiratory rate; tidal and minute volume; esophageal pressure; inspiratory and expiratory time; peak inspiratory and peak expiratory flow; expiratory resistance; dynamic compliance.
- Lung volume diffusion capacity and pressure-volume relationships: vital and total lung capacity; end expiratory and residual volume; diffusion capacity of carbon monoxide; chord slope, and peak compliance.
- Forced expiration measurements: forced vital capacity; forced expiratory volume at 50, 100, 200, and 400 mseconds; peak expiratory flow; volume at peak expiratory flow; mean mid-expiratory flow; flow at 75%, 50%, 25%, and 10% of forced vital capacity.
- Pulmonary lavage analyses: total, viable, and differential cell counts; protein concentration
Sacrifice and pathology:
Method of sacrifice: CO2 asphyxiation.

GROSS PATHOLOGY: Yes.
- Necropsy was performed at study end on all core study animals.
- Organs weighed were the heart, right kidney, liver, lung, right testis, and thymus.

HISTOPATHOLOGY: Yes.
- Complete histopathology was performed on control, 8 and 16 mg/m3 rats. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gallbladder (mice only), heart and aorta, large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum, and ileum), kidney, larynx, liver, lung and mainstem bronchi, lymph nodes (mandibular, mediastinal, mesenteric, and bronchial), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder and uterus.
- The lung and nose of rats in all remaining exposure groups were also examined.
Other examinations:
- Before the studies began, five male and five female rats were randomly selected for parasite evaluation and gross observation for evidence of disease.
- At the end of the studies, serologic analyses were performed on five male and five female sentinel rats using the protocols of the NTP Sentinel Animal Program.
Statistics:
Survival Analyses:
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. Animals found dead of other than natural causes were censored from the survival analyses; animals dying from natural causes were not censored. Statistical analyses for possible doserelated effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.

Analysis of Continuous Variables:
Two approaches were employed to assess the significance of pairwise comparisons between exposed and control groups in the analysis of continuous variables. Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Hematology, clinical chemistry, urinalysis, urine concentrating ability, cardiopulmonary, immunotoxicologic, cell proliferation, tissue concentrations, spermatid, and epididymal spermatozoal data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test). Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973). Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across exposure concentrations. (for more information see publication)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- In the highest dose groups (16 mg/m3) abnormal breathing, thinness, lethargy, abnormal posture and ruffled fur were observed
- Some rats in the 16 mg/m3 groups had diarrhea and nasal/eye discharge.
Mortality:
mortality observed, treatment-related
Description (incidence):
- 7 males and 3 females exposed to 16 mg/m3 died during the study
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Final mean body weights and body weight gains of males exposed to 4 mg/m3 or greater and of females exposed to 16 mg/m3 were significantly less than those of the chamber controls
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- Microcytic erytrocytosis was observed in males and females
- Exposure of rats to vanadium pentoxide affected the circulating red cell mass
- In general, the erythrocyte counts reflected similar and proportional, but not significant, decreases.
- There were exposure concentration-related decreases in the mean cell volumes and mean cell hemoglobin values on day 23, which are consistent with a developing erythrocyte microcytosis.
- The mean cell hemoglobin concentration was minimally decreased in 16 mg/m3 females
- Microscopic evaluation of the red blood cell morphology detected increased polychromasia, anisochromia, and hypochromia in the 16 mg/m3 groups
- At week 13, there was evidence of an exposure-related leukopenia, demonstrated by decreases in leukocyte counts in 16 mg/m3 males and females; leukopenia appeared to be related to decreased lymphocyte counts. This alteration in lymphocyte counts suggests a physiological response and would be consistent with a stress-related corticosteroid- induced lymphopenia. Rats are considered a steroid-sensitive species and corticosteroid-induced lymphopenia may be related to lympholysis in blood and altered distribution (Jain, 1986).
- The 16 mg/m3 males and females demonstrated microscopic evidence of lymphoid depletion in numerous lymphoid organs. Since there was an exceptional effect on body weights of 16 mg/m3 rats, it is possible that these animals were under significant stress that then lead to increased endogenous corticosteroid concentrations and subsequent decreases in tissue and blood lymphocytes.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Description (incidence and severity):
effects not toxicologically relevant
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- Absolute and relative lung weights were significantly higher at and above 4 mg/m3 in males and females than for the chamber controls as were the relative lung weights of 2 mg/m3 males
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Lungs:
- Incidence if several nonneoplastic lesions of the lung and nose were increased in animals exposed to 2 mg/m3 or greater
- There were significant increases in the incidences of epithelial hyperplasia of the lung in males and females exposed to 2 mg/m3 or greater.The epithelial hyperplasia occurred in the distal airways and associated alveolar ducts and alveoli. In the distal airways, the epithelium was composed of ciliated cuboidal to columnar cells crowded together in multiple layers, sometimes forming folds and small papillary projections. More distal and including the alveolar ducts and alveoli, the epithelium tended to be a single layer. The epithelial cells were larger and rounded and appeared morphologically similar to bronchioles. Goblet cells sometimes occurred within the epithelium of some of the smaller airways. Alveoli often contained one or two cells that were very large and occasionally binucleate.
- The incidences of inflammation or fibrosis were significantly increased in males exposed to 2 mg/m3 or greater and females exposed to 4 mg/m3 or greater.
- In one female exposed to 16 mg/m3, a single focus of squamous metaplasia was observed within an area of hyperplasia.
- Exudate of the bronchiole was diagnosed in many males and females exposed to 8 or 16 mg/m3 and was characterized by a mucoid material with admixed inflammatory cells within the lumens of some of the hyperplastic airways. The inflammation was characterized by accumulations of alveolar macrophages with abundant foamy cytoplasm in the alveoli adjacent to airways lined by hyperplastic epithelium. Mononuclear cells were also observed within the interstitium.
- Foci of fibrosis were found in the same areas and sometimes extended into surrounding alveoli. These foci were irregularly shaped and consisted of loosely arranged, delicate collagen fibers, fibroblasts, and mononuclear cells.

Nose:
- The incidences of hyperplasia and metaplasia of the nasal respiratory epithelium were significantly increased in males exposed to 8 or 16 mg/m3 and in females exposed to 4 mg/m3 or greater. There were significantly increased incidences of inflammation of the nose in males and females exposed to 16 mg/m3. The hyperplasia and metaplasia involved the respiratory epithelium covering the ventral portion of the nasal septum, the vomeronasal organ, and, to a lesser extent, the ventral lateral walls of the anterior portion of the nasal cavity. There were cellular crowding and epithelium thickening (hyperplasia) in some areas with loss of goblet cells and one or more layers of flattened nonkeratinizing squamous epithelium (metaplasia) in others.
- Inflammation was characterized by an infiltrate primarily of lymphocytes and lesser numbers of neutrophils within the affected mucosa and subjacent submucosa. Occasionally, neutrophils predominated, and an exudate was present.

Other:
- Depletion of lymphocytes in the spleen, thymus, and lymph nodes, atrophy of metaphyseal bone of the femur, and atrophy of the secondary reproductive organs were observed in 16 mg/m3 males and females and hypospermia of the testis and atypical cells of the epididymis were observed in 16 mg/m3 males. These lesions may have been associated with the marked body weight loss and general debilitation of these rats.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
- Significant exposure-related changes in pulmonary function were observed in male and female rats exposed to 4, 8, or 16 mg/m3; these results indicate that more restrictive lesions were present in groups exposed to 4 mg/m3 or greater, evidenced by reduced lung compliance, changes in breathing measurements, impaired capacity to diffuse carbon monoxide, reduced static and dynamic lung volumes, and exaggerated flows.
- Pulmonary function changes indicate an obstructive disease in the 16 mg/m3 groups, evidenced by changes in breathing mechanics, static lung volumes, and forced expiratory maneuvers. Expiratory resistance, an indicator of bronchoconstriction, and end expiratory and residual volume were increased, while dynamic lung volume was decreased.
- Together, the pulmonary function changes indicate that a restrictive disease was present in male and female rats exposed to 4 mg/m3 or greater, while an obstructive lung disease may have been present only in the 16 mg/m3 groups.
- The pulmonary lavage data indicate an inflammatory response in the lungs of exposed rats. Exposure concentration-related increases were observed in the total numbers of cells, lymphocytes, neutrophils, and protein recovered in pulmonary lavage fluid from rats exposed to vanadium pentoxide at concentrations up to 8 mg/m3.
Details on results:
CLINICAL CHEMISTRY
- At all time points, evidence of a hepatocellular effect was demonstrated by increases in serum alanine aminotransferase activities in 8 and 16 mg/m3 males and females. However, sorbitol dehydrogenase activities, another marker of hepatocellular injury or leakage, were not affected, and there were no histopathologic liver changes consistent with hepatocellular injury. This suggests that the increased alanine aminotransferase activities were not related to hepatocellular injury. Studies have shown that corticosteroids can induce increases in liver alanine animotransferase activities (Rosen, 1959; Rosen et al., 1959). Thus, stress-related corticosteroid-induced increases in liver alanine aminotransferase activities may, in part, help explain increased serum alanine aminotransferase activities.
- At all time points, there were generally exposure concentration-related decreases in serum alkaline phosphatase activities and total protein and albumin concentrations in males and females. At week 13, urea nitrogen concentrations were increased in 16 mg/m3 males and females, suggesting a possible effect on renal clearance. However, creatinine concentrations, another marker of renal clearance, were minimally decreased on day 23 in 8 mg/m3 males and at week 13 in 16 mg/m3 males and females; these decreases would be consistent with the decreased body weights observed in these groups. Since alkaline phosphatase activity and total protein, albumin, and urea nitrogen concentrations can be affected by altered nutritional status, the changes in these variables may have been related secondarily to body weight decreases and altered food intake.
- Significant increases and decreases in bile acid concentrations and creatine kinase activities in various exposure groups at various time points were not considered to be toxicologically relevant.

URINALYSIS
- After 12 (females) or 13 (males) weeks of exposure, the baseline (water-replete) overnight urine collection demonstrated decreased urine volumes and increased urine specific gravities in the 8 mg/m3 male and 16 mg/m3 female groups, suggesting these animals were able to concentrate their urine; the 16 mg/m3 male group was not tested. These findings also suggest that these exposure groups may have been in a partially dehydrated state prior to the water deprivation studies.
- The 4-hour urine collection following a 16-hour water deprivation period demonstrated no differences in urine volume or specific gravity. This suggests that animals in all exposed groups maintained their ability to concentrate urine.
- For baseline and water deprivation test samples, microscopic evaluation of the urine demonstrated slight increases in formed elements in 8 mg/m3 males (baseline: casts, epithelial cells, erythrocytes, and leukocytes; water-deprived: casts) and 16 mg/m3 females (baseline and water deprived: leukocytes) (data not presented). While changes in formed urine elements can be indicative of various renal effects, the alterations in this study were not excessive and possibly reflected the hydration status of the animals; they were not considered to be toxicologically relevant.

ORGAN WEIGHTS
- Other organ weight differences were considered to be related to body weight decreases

GROSS PATHOLOGY
- The carcasses of males and females exposed to 16 mg/m3 were very thin, and the spleens and thymuses appeared disproportionately small
- Lungs of 4 mg/m3 or greater males and 8 and 16 mg/m3 females varied from red to pale or mottled.
Dose descriptor:
NOAEC
Remarks:
local
Effect level:
1 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Critical effects observed:
not specified

Based on the incidences and severities of respiratory lesions and increased lung weights in male and female rats, concentrations

of 4 mg/m3 or greater were considered to be too high for use in a 2-year study. The exposure concentrations selected for the 2-year inhalation study in rats were 0.5, 1, and 2 mg/m3.

Conclusions:
The 16 mg/m3 concentration was lethal to several rats and body weights were severely reduced in this exposure group. The respiratory tract was clearly the primary site of toxicity in rats exposed to vanadium pentoxide. The respiratory effects were more intense with increased exposure time, as indicated by increased lung weights and a greater spectrum and increased severity of proliferative and inflammatory lesions in the lungs of most exposed rats. Alveolar/bronchiolar epithelial hyperplasia was present in all rats exposed to 2 mg/m3 or greater. Minimal to moderate fibrosis of the lung occurred in rats exposed to 2 mg/m3 or greater. The no-observed-adverse-effect concentration (NOAEC) for lungs was 1 mg/m3 in rats. Pulmonary function and lung lavage analyses were performed. Taken together, hyperplastic and inflammatory lesions, the presence of exudate in the bronchioles, and lavage fluid analysis supported the conclusion from the pulmonary function tests that indicated restrictive lung disease in rats exposed to 4 mg/m3 or greater.
Reason / purpose:
reference to same study
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1990-09-20 (first exposure) to 1990-12-21/22 (necropsy date)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well reported study. Test procedure in accordance with generally accepted scientific standards and described in sufficient detail.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 10 male and 10 female mice were exposed to particulate aerosols of vanadium pentoxide at concentrations of 0, 1, 2, 4, 8, or 16 mg/m3 by inhalation, 6 hours per day, 5 days per week for 3 months. The 3-month studies were conducted to evaluate the cumulative toxic effects of repeated exposure to vanadium pentoxide and to determine the appropriate exposure concentrations to be used in the 2-year studies.
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: average age: 6 or 7 weeks old on the first day of the study (4 weeks old on receipt)
- Weight at study initiation: range of mean body weights in the exposure groups: 25-26 g (males) and 20-21 g (females)
- Housing: housed individually; stainless steel wire mesh (Lab Products, Inc., Maywood, NJ), changed weekly
- Diet: ad libitum, except during exposure periods; NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA)
- Water: ad libitum; tap water (City of Chicago municipal supply) via automatic watering system
- Acclimation period: quarantined for 10 or 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 23.9± ca.2°C (75° ± 3° F)
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day

IN-LIFE DATES: From: 1990-09-20 (first exposure) to 1990-12-21/22 (necropsy date)
- No further information on test material was stated.
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: MMAD = 1.0-1.3 µm, GSD = 2.3-2.8
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- For the 16-day and 3-month studies, vanadium pentoxide aerosol generation was based on the principle of pneumatic dispersion and consisted of two major components: a screw feeder (Model 310, Accurate, White Water, WI) that metered vanadium pentoxide powder at a constant rate and a Jet-O-Mizer jetmill (Fluid Energy Corp., Harfield, PA) that used compressed air to disperse the metered powder and form the aerosol.
- Aerosol leaving the jetmill passed through a one-stage impactor and a vertical elutriator to eliminate or deagglomerate the large particles before entering a plenum and manifold distribution system. The aerosol delivery system consisted of three holding chambers that diluted the aerosol in three stages. A metered amount of diluted aerosol was removed and mixed with conditioned air at the inlet to each exposure chamber to achieve the appropriate exposure concentration. The electrical charge buildup on the aerosol particles was neutralized by mixing the aerosol with high concentrations of bipolar ions, which were generated using a Pulse Gun (Static Control Services, Palm Springs, CA) air nozzle. For the 3-month studies, a transvector air pump was installed at the aerosol inlet to each exposure chamber to provide additional control of the aerosol flow rate and improve stability of the chamber concentration.
- The stainless-steel inhalation exposure chambers (Lab Products, Inc., Maywood NJ), were designed so that uniform aerosol concentrations could be maintained throughout the chambers when catch pans were in place. The total active mixing volume of each chamber was 1.7 m³.

CHAMBER ATMOSPHERE CHARACTERIZATION
- The particle size distribution in each chamber was determined prior to the start of all studies, during the first week of the 16-day and 3-month studies, during the first 2 weeks of the 2-year studies, and monthly during the 3-month and 2-year studies.
- For the 16-day and 3-month studies, a 10-stage Quartz Crystal Microbalance-based cascade impactor was used to separate the aerosol particles into sequential size ranges; the mass median aerodynamic diameter was calculated from the corresponding mass fraction of particles at each stage.

OTHER
- The uniformity of aerosol concentration in the inhalation exposure chambers without animals was evaluated before each of the studies began; concentration uniformity with animals present in the chambers was also measured. During the 16-day and 3-month studies, minor excursions in chamber uniformity values were observed in one or more exposure chambers, but these excursions had no impact on the studies. Chamber concentration uniformity was acceptable throughout the 16-day special studies and 2-year studies.
- The stability of vanadium pentoxide in the exposure system was tested with XRD analysis. XRD analyses indicated no detectable build-up of degradation products at a detection limit of approximately 1%.



Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- During all studies, chamber aerosol concentrations were monitored with real-time aerosol monitors (RAMs) that used a pulsed-light-emitting diode in combination with a silicon detector to sense light scattered over a forward angular range of 45° to 95° by particles traversing the sensing volume. The instruments respond to particles 0.1 to 20 μm in diameter.
- During the 16-day and 3-month studies, an individual monitor was used for each exposure chamber. The voltage output of the online monitors was read and recorded, and the calibration curve was applied to the voltages measured by the RAM to convert the measured voltages to exposure chamber concentrations.
- Each RAM was calibrated daily during the 16-day and 3-month studies by correlating the measured voltage with vanadium pentoxide concentrations determined by gravimetric analysis of glass fiber filters and one to two times per week during the 2-year studies by ICP/AES or ICP/mass spectrometry analysis of Pallflex® TX40H120WW glass fiber filters.
Duration of treatment / exposure:
3 months (91-92 days)
Frequency of treatment:
6 hours per day, 5 days per week
Remarks:
Doses / Concentrations:
1 mg/m³ V2O5
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2 mg/m³ V2O5
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
4 mg/m³ V2O5
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
8 mg/m³ V2O5
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
16 mg/m³ V2O5
Basis:
nominal conc.
No. of animals per sex per dose:
groups of 10 male and 10 female mice
Control animals:
yes
Details on study design:
- Dose selection rationale: based on decreased survival in the 32 mg/m3 males and body weight decreases in 32 mg/m3 males and females, an exposure concentration of 32 mg/m3 was considered too high for use in a 3-month study. Therefore, the exposure concentrations selected for the 3-month inhalation study in mice were 0, 1, 2, 4, 8, and 16 mg/m3.
- Rationale for animal assignment (if not random): randomly into groups of approximately equal initial mean body weights.
- No further information on test material was stated.
Positive control:
not stated
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were observed twice daily; clinical findings were recorded weekly

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: body weights were controlled initially, weekly and at study end

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

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 data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No



Sacrifice and pathology:
Method of sacrifice CO2 asphyxiation.

GROSS PATHOLOGY: Yes
- Necropsy was performed at study end on all core study animals.
- Organs weighed were the heart, right kidney, liver, lung, right testis, and thymus.

HISTOPATHOLOGY: Yes
- Complete histopathology was performed on control, and 16 mg/m3 mice. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gallbladder (mice only), heart and aorta, large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum, and ileum), kidney, larynx, liver, lung and mainstem bronchi, lymph nodes (mandibular, mediastinal, mesenteric, and bronchial), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus.
- The lungs of mice in all remaining exposure groups and the thymus in 8 mg/m3 mice were also examined.
Other examinations:
- Before the studies began, five male and five female mice were randomly selected for parasite evaluation and gross observation for evidence of disease.
- At the end of the studies, serologic analyses were performed on five male and five female sentinel mice using the protocols of the NTP Sentinel Animal Program.
Statistics:
Survival Analyses:
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. Animals found dead of other than natural causes were censored from the survival analyses; animals dying from natural causes were not censored. Statistical analyses for possible doserelated effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.

Analysis of Continuous Variables:
Two approaches were employed to assess the significance of pairwise comparisons between exposed and control groups in the analysis of continuous variables. Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Hematology, clinical chemistry, urinalysis, urine concentrating ability, cardiopulmonary, immunotoxicologic, cell proliferation, tissue concentrations, spermatid, and epididymal spermatozoal data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test). Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973). Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across exposure concentrations. (for more information see publication)
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
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 specified
Details on results:
CLINICAL SIGNS AND MORTALITY
- One male exposed to 16 mg/m³ died before the end of the study

BODY WEIGHT AND WEIGHT GAIN
- Mean body weights of 8 and 16 mg/m³ males and 4 mg/m³ or greater females were significantly less than those of the chamber controls

ORGAN WEIGHTS
- Absolute and relative lung weights of males and females exposed to 4 mg/m³ or greater were significantly greater than those of the chamber controls
- The absolute lung weight was also significantly increased in males exposed to 2 mg/m3
- Other organ weight changes were considered related to body weight decreases

GROSS PATHOLOGY
- Gross findings were observed in males and females exposed to 8 or 16 mg/m3 and included lungs that were pale or contained white or red (females) foci; the lungs of males in these groups were sometimes gray or mottled

HISTOPATHOLOGY: NON-NEOPLASTIC
- Some mice exposed to 2 or 4 mg/m³ and all mice at and above 8 mg/m3 had inflammation and epithelial hyperplasia of the lung. The severities of these lesions generally increased with increasing exposure concentration
Dose descriptor:
NOAEC
Remarks:
local
Effect level:
1 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: increased absolute lung weights and epithelial hyperplasia and inflammation in lungs at 2 mg/m3
Critical effects observed:
not specified

Based on reduced body weight gain of 8 and 16 mg/m3 males and females and increased incidences of inflammation and epithelial hyperplasia of the lung in 8 and 16 mg/m3 males and females, exposure concentrations greater than 4 mg/m3 were considered too high for use in a 2-year study. The exposure concentrations selected for the 2-year inhalation study in mice were 1, 2, and 4 mg/m3.

Conclusions:
The 16 mg/m3 concentration was lethal to one male mouse. The respiratory tract was clearly the primary site of toxicity in mice exposed to vanadium pentoxide. The respiratory effects were more intense with increased exposure time, as indicated by increased lung weights and a greater spectrum and increased severity of proliferative and inflammatory lesions in the lungs of most exposed mice. Alveolar/bronchiolar epithelial hyperplasia was present in mice exposed at and above 2 mg/m3. The no-observed-adverse-effect-concentration was 1 mg/m3 for effects in lungs.
Reason / purpose:
reference to same study
Reference
Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1997-01-13 (first exposure) to 1999-01-11/15 (necropsy date)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well reported study. Test procedure in accordance with generally accepted scientific standards and described in sufficient detail.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 50 male and 50 female B6C3F1 mice (approx. 6-7 weeks old) were exposed to V2O5 aerosols at concentrations of 0, 1, 2 or 4 mg/m3 by inhalation, 6 hours/d, 5 d/wk, for 104 weeks. The body weight was controlled initially and body weight and clinical finding were recorded every 4 weeks (until week 89) and every 2 weeks from week 92 on. Animals were observed twice daily. Necropsy was performed at study end.
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: average age: 6 or 7 weeks old on the first day of the study
- Weight at study initiation: average body weight for week 1: 24.0-24.4 g (males) and 19.6-19.9 g (females)
- Housing: housed individually; stainless steel wire bottom (Hazleton Systems, Inc., Aberdeen, MD); cages and racks were rotated weekly.
- Diet: ad libitum, except during exposure periods; NTP-2000 pelleted diet (Zeigler Brothers, Inc., Gardeners, PA), changed weekly
- Water: ad libitum; tap water (Richland, WA, municipal supply water used) via automatic watering system
- Acclimation period: quarantined for 16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 23.9± ca.2°C (75° ± 3° F)
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day

IN-LIFE DATES: From: 1997-01-13 (first exposure) to 1999-01-11/15 (necropsy date)
- No further information on test material was stated.
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: MMAD = 1.0-1.3 µm, GSD = 2.3-2.8
Details on inhalation exposure:
- The generation and delivery system used in the 16-day special studies and the 2-year studies consisted of a linear dust feeder, a particle attrition chamber, and an aerosol distribution system. The linear dust feeder, a slide-bar dust-metering device, was composed of a shuttle bar, body, outlet port, and hopper. As the compressed-air-driven shuttle bar slid back and forth during generation, the metering port aligned with the hopper, which served as a reservoir for the bulk chemical, and was filled with a small amount of vanadium pentoxide powder. As the shuttle bar slid to the dispersing position, the metering port aligned with a compressed-air port in the body and a puff of air from this port dispersed the vanadium pentoxide into the particle attrition chamber. Generator output was regulated by adjusting the cadence of the shuttle bar. The particle attrition chamber used low fluid energy from an air jet tangential to the chamber to deagglomerate the vanadium pentoxide particles. After deagglomeration, the particles were swept into a classification zone where smaller particles exited to the distribution line; larger particles were thrown to the perimeter of the classifier by centrifugal force and were reentrained into the impacting air jet, and the process was repeated until the particles were sufficiently deagglomerated. The aerosol passed through the distribution lines to the exposure chambers. A pneumatic pump designed by the study laboratory was located at each chamber inlet and drew aerosol from the distribution line into the chamber inlet, where it was diluted with conditioned air to the appropriate concentration. Flow through the distribution line was controlled by Air-Vac pumps (Air-Vac Engineering, Milford, CT), and pressure was monitored by photohelic differential pressure gauges (Dwyer Instruments, Inc., Michigan City, IN).
- The Stainless steel chambers (Lab Products, Inc., Harford Systems Division, Aberdeen, MD), were designed so that uniform aerosol concentrations could be maintained throughout the chambers when catch pans were in place. The total active mixing volume of each chamber was 1.7 m³.

CHAMBER ATMOSPHERE CHARACTERIZATION
- The particle size distribution in each chamber was determined prior to the start of all studies, during the first week of the 16-day and 3-month studies, during the first 2 weeks of the 2-year studies, and monthly during the 3-month and 2-year studies.
- For the 16-day special studies and the 2-year studies, a Mercer-style seven-stage impactor was used. The stages (glass coverslips lightly sprayed with silicon) were analyzed by ICP/AES, and the relative mass collected on each stage was analyzed by probit analysis.

OTHER
- The uniformity of aerosol concentration in the inhalation exposure chambers without animals was evaluated before each of the studies began; concentration uniformity with animals present in the chambers was also measured. During the 16-day and 3-month studies, minor excursions in chamber uniformity values were observed in one or more exposure chambers, but these excursions had no impact on the studies. Chamber concentration uniformity was acceptable throughout the 16-day special studies and 2-year studies.
- The stability of vanadium pentoxide in the exposure system was tested with XRD analysis. XRD analyses indicated no detectable build-up of degradation products at a detection limit of approximately 1%.



Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- During all studies, chamber aerosol concentrations were monitored with real-time aerosol monitors (RAMs) that used a pulsed-light-emitting diode in combination with a silicon detector to sense light scattered over a forward angular range of 45° to 95° by particles traversing the sensing volume. The instruments respond to particles 0.1 to 20 μm in diameter.
- For the 16-day special studies and the 2-year studies, the sampling system consisted of a valve that multiplexed each RAM to two or three exposure chambers and to a HEPA filter and/or the control chamber or room; selection of sampling streams and data acquisition from each RAM was remotely controlled by a computer. Equations for calibration curves were stored in the computers and were used to convert the measured voltages to exposure concentrations.
- Each RAM was calibrated daily during the 16-day and 3-month studies by correlating the measured voltage with vanadium pentoxide concentrations determined by gravimetric analysis of glass fiber filters and one to two times per week during the 2-year studies by ICP/AES or ICP/mass spectrometry analysis of Pallflex® TX40H120WW glass fiber filters.
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
6 hours per day, 5 days per week
Remarks:
Doses / Concentrations:
1 mg/m³ V2O5
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2 mg/m³ V2O5
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
4 mg/m³ V2O5
Basis:
nominal conc.
No. of animals per sex per dose:
core study: 50 males and 50 females per dose
tissue burden analyses: 40 female mice per exposed group; separate control group of 15 female mice was used as chamber controls
Control animals:
yes
Details on study design:
- Dose selection rationale: the highest exposure concentration (4 mg/m3) selected for mice was based on reduced body weight gains of 8 and 16 mg/m3 males and females, increased lung weights of 4 mg/m3 or greater males and females, increased incidences of epithelial hyperplasia, and increased incidences and severities of inflammatory lesions of the lungs in mice exposed to 8 mg/m3 versus those exposed to 4 mg/m3 in the 90-day study.
- Rationale for animal assignment (if not random): randomly into groups of approximately equal initial mean body weights.
- No further information on test material was stated.
Positive control:
not stated
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Clinical findings were recorded every 4 weeks from week 5 through 89 and every 2 weeks from week 92 to the end of the study

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed at the beginning of the studies. Body weights were recorded every 4 weeks from week 5 through 89 and every 2 weeks from week 92 to the end of the study

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

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 data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
Tissue Burden Studies:
- Lungs and whole blood were collected from five mice in each exposure group sacrificed on day 1, 5, 12, 26, 54, 171, 362, or 535. Groups of five chamber control animals were bled at each of these time points and returned to their chambers and used for subsequent bleedings. The lungs and associated tissues were removed from exposed animals; after the left and right lung were prepared for histopathologic examination, and the right lung was digested to measure vanadium concentration. Vanadium concentrations in blood and right lung nitric acid digests were measured. Calculations of lung deposition and clearance parameters were performed. Blood vanadium concentration was measured in all animals at all time points after day 12.
Sacrifice and pathology:
Method of Sacrifice: CO2 asphyxiation.

PATHOLOGY: Yes
- Complete necropsies were performed on all core study mice.
- All organs and tissues were examined for grossly visible lesions, and all major tissues were prepared for microscopic examination.

HISTOPATHOLOGY: Yes
- Complete histopathology was performed on all core study animals. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gallbladder (mice only), heart and aorta, large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum, and ileum), kidney, larynx, liver, lung and mainstem bronchi, lymph nodes (mandibular, mediastinal, mesenteric, and bronchial), mammary gland (except male mice), nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder and uterus.
Other examinations:
Five male and five female mice were randomly selected for parasite evaluation and gross observation of disease.
Statistics:
Survival Analyses:
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. Animals found dead of other than natural causes were censored from the survival analyses; animals dying from natural causes were not censored. Statistical analyses for possible doserelated effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.

Analysis of Continuous Variables:
Two approaches were employed to assess the significance of pairwise comparisons between exposed and control groups in the analysis of continuous variables. Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Hematology, clinical chemistry, urinalysis, urine concentrating ability, cardiopulmonary, immunotoxicologic, cell proliferation, tissue concentrations, spermatid, and epididymal spermatozoal data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test). Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973). Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across exposure concentrations. (for more information see publication)
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY
- Survival of the highest dosed males (4 mg/m³) was significantly less than controls
- Abnormal breathing was observed particularly those exposed to 2 or 4 mg/m³
- Many mice exposed to vanadium pentoxide were thin

BODY WEIGHT AND WEIGHT GAIN
-Mean body weight of all females and highest dosed males was below the controls; and mean body weights of males exposed to 2 mg/m3 were less from week 85 to the end of the study

HISTOPATHOLOGY: NON-NEOPLASTIC
- Non-neoplastic lesions occurred in respiratory system (lung, larynx, and nose) of males and females, and the severities of these lesions generally increased with increasing exposure concentration.
-Bronchial lymph node hyperplasia was present in most of the exposed females.

LUNG:
- There were significantly increased incidences of alveolar epithelial hyperplasia and bronchiolar epithelial hyperplasia in the lungs of exposed male and female mice. Incidences of chronic inflammation and histiocytic cellular infiltrate were significantly increased in exposed groups of mice, and the incidences of interstitial fibrosis were increased in mice exposed to 2 or 4 mg/m3.
- Males: alveolar epithelium, hyperplasia (3/50, 41/50, 49/50, 50/50); bronchiole, epithelium, hyperplasia (0/50, 15/50, 37/50, 46/50); inflammation chronic (6/50, 42/50, 45/50, 47/50); alveolus, infiltration cellular, histiocyte (10/50, 36/50, 45/50, 49/50); interstitial fibrosis (1/50, 6/50, 9/50, 12/50)
- Females: alveolar epithelium, hyperplasia (0/50, 31/50, 38/50, 50/50); bronchiole, epithelium, hyperplasia (0/50, 12/50, 34/50, 48/50); inflammation chronic (4/50, 37/50, 39/50, 49/50); alveolus, infiltration cellular, histiocyte (0/50, 34/50, 35/50, 45/50); interstitial fibrosis (0/50, 1/50, 4/50,
8/50)
- The hyperplasia was essentially a diffuse change with proliferation of epithelium in the distal terminal bronchioles and immediately associated alveolar ducts and alveoli. Normally flattened epithelium was replaced with cuboidal epithelium. The hyperplasia of the alveolar epithelium was pronounced and increased in severity with increasing exposure concentration, while the hyperplasia of the distal bronchioles was minimal to mild with slight increases in severity in mice exposed to 4 mg/m3. The changes in mice were similar to those observed in rats but were not as pronounced.
- The inflammatory lesions were primarily minimal to mild and consisted of interstitial, perivascular, and peribronchiolar infiltrates of mostly mononuclear inflammatory cells (mostly lymphocytes) that were occasionally present within alveoli. The most prominent histiocytic infiltrate occurred within alveoli in close proximity to alveolar/bronchiolar neoplasms, particularly carcinomas.

LARYNX:
- There were significantly increased incidences of minimal squamous metaplasia of the respiratory epithelium of the epiglottis in exposed groups.
- Males: respiratory epithelium, epiglottis, metaplasia, squamous (2/49, 45/50, 41/48, 41/50)
- Females: respiratory epithelium, epiglottis,metaplasia, squamous (0/50, 39/50, 45/49, 44/50)
- Squamous metaplasia was diagnosed when the ciliated cells were replaced by one to three layers of flattened squamous epithelium. This change commonly occurs in mice in NTP inhalation studies and represents a common response to laryngeal injury.

NOSE:
- There were increased incidences of minimal to mild suppurative inflammation of the nose in males and females exposed to 2 or 4 mg/m3.
- Males: inflammation suppurative (16/50, 11/50, 32/50, 23/50); olfactory epithelium, degeneration, hyaline (1/50, 7/50, 23/50, 30/50); respiratoryepithelium, degeneration, hyaline (8/50, 22/50, 38/50, 41/50); respiratory epithelium, metaplasia, squamous (0/50, 6/50, 6/50, 2/50)
- Females: inflammation suppurative (19/50, 14/50, 32/50, 30/50); olfactory epithelium, atrophy (2/50, 8/50, 5/50, 14/50); olfactory epithelium, degeneration, hyaline (11/50, 23/50, 34/50, 48/50); respiratory epithelium, degeneration, hyaline (35/50, 39/50, 46/50, 50/50); respiratory epithelium, metaplasia, squamous (0/50, 3/50, 7/50, 8/50); respiratory epithelium, necrosis (0/50, 0/50, 1/50, 7/50)
- The inflammation consisted of focal aggregates of few to moderate numbers of neutrophils generally subjacent to the epithelium of the turbinates, septum, or lateral wall of the anterior nose. In the more severe cases (predominantly in females exposed to 4 mg/m3), a short segment of the overlying epithelium was ulcerated (necrosis). Similarly, in some males and females, the overlying respiratory epithelium was replaced by one or more layers of flattened epithelium (squamous metaplasia), and the incidences were marginally increased in some exposed groups of mice.
- The majority of the olfactory epithelium covers the turbinates in the distal portion of the nose. There were marginal but significant increases in the incidences of atrophy of this epithelium in females exposed to 1 or 4 mg/m3.

BRONCIAL LYMPH NODE
- There were significant increases in the incidences of hyperplasia of the bronchial lymph node in exposed groups of females, and while not significant, a positive trend in the incidences of this lesion also occurred in males.
- Females: hyperplasia (3/39, 13/40, 14/45, 20/41)

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
LUNG:
- The incidences of alveolar/bronchiolar carcinoma and alveolar/bronchiolar adenoma or carcinoma (combined) were significantly increased in all groups of exposed male and female mice compared to controls
- Males: alveolar/bronchiolar adenoma (13/50, 16/50, 26/50, 15/50); alveolar/ bronchiolar carcinoma (12/50, 29/50, 30/50, 35/50); alveolar/ bronchiolar adenoma or carcinoma (22/50, 42/50, 43/50, 43/50)
- Females: alveolar/bronchiolar adenoma (1/50, 17/50, 23/50, 19/50); alveolar/ bronchiolar carcinoma (0/50, 23/50, 18/50, 22/50); alveolar/ bronchiolar adenoma or carcinoma (1/50, 32/50, 35/50, 32/50)
- The incidences of alveolar/bronchiolar adenoma were significantly increased in males exposed to 2 mg/m3 and in all groups of exposed females.
- These increased incidences exceeded the historical ranges for controls.

SPLEEN:
- There was a positive trend in the incidences of hemangiosarcoma of the spleen in male mice (chamber control, 0/50; 1 mg/m3, 0/50; 2 mg/m3, 0/50; 4 mg/m3, 3/50).
- However, the incidence in the 4 mg/m3 group was within the historical range in controls.
- In addition, the incidences of hemangioma or hemangiosarcoma (combined) in all organs were not significant. The marginal increase in the incidence of hemangiosarcoma of the spleen was not considered related to vanadium pentoxide exposure.

OTHER FINDINGS
TISSUE BURDEN STUDIES:
- Histopathology: lung lesions were identified early, and were more severe in females exposed to higher concentrations and progressed with time. Minimal bronchiolar epithelial hyperplasia and interstitial inflammation were initially observed in the 4 mg/m3 group on day 5. Bronchiolar epithelial hyperplasia was characterized by slight piling up of cells, some nuclear pleomorphism, and occasional karyorrhectic cells. On day 12, exposure-related lesions were evident in 2 mg/m3 females, while alveolar epithelial hyperplasia was more prominent in the 4 mg/m3 group. Lesions were first observed in the 1 mg/m3 group on day 54, and lesion severities in the 2 and 4 mg/m3 groups appeared to be increased on day 54. On day 362, lesion severity in lungs of females exposed to 4 mg/m3 was noticeably increased, and bronchiolar hyperplasia included more nuclear pleomorphism. On day 535, mice exposed to 2 mg/m3 had hyperplastic alveolar epithelial lesions and, similar to females exposed to 4 mg/m3, the hyperplasia was oriented around the terminal bronchiolar/alveolar duct. Also on day 535, lung nodules/masses were observed. While some were confirmed as neoplasms, many were in the right lung lobe and thus were not confirmed histopathologically. Similar lesions, although more severe, were observed in the core group of animals at 2 years.
- Lung burden data appeared proportional to exposure concentration in mice. Rats removed vanadium from the lungs much slower than mice, with clearance half-times of vanadium lung burdens approximately six to nine fold longer in rats than in mice at comparable exposure concentrations. In rats and mice, lung clearance half-times were considerably longer than those observed in the 16-day special studies.
- The maximum lung burdens occurred between days 26-54 in mice. The lung burdens appeared to reach steady state at the lowest exposure concentrations (1 mg/m3). A decline in lung burdens was observed. The retention of vanadium in the lungs at 18 months was 2% to 3% in mice. The total lung doses for mice exposed to 1, 2, or 4 mg/m3 were estimated to be 153, 162, and 225 μg vanadium, respectively. There was little difference in the total lung dose for mice, especially between the 1 and 2 mg/m3 groups.
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
1 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: non-neoplastic changes (epithelial hyperplasia, squamous metaplasia, chronic inflammation, degeneration) in the respiratory system (lung, larynx, and nose) of male and female mice
Critical effects observed:
not specified

Chamber concentration uniformity was acceptable throughout the 16-day special studies and 2-year studies.

Conclusions:
The survival rate 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.
Reason / purpose:
reference to same study
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no data available
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
not applicable
Species / strain / cell type:
S. typhimurium TA 97
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
TA102, TA1535 and TA 97: 0, 0.03, 0.1, 0.3, 1.0, 3.0, 6.0, 10.0 and 33.0 µg/plate
TA100 and TA 98: 0, 0.1, 0.3, 1.0, 3.0, 6.0, 10.0, 33.0, 100.0 and 333.0 µg/plate
Vehicle / solvent:
No vehicle is reported, but in the results (summary table) of the reference an evidence is given that a vehicle was used: "0 μg/plate was the solvent control."
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
mitomycin C
other: 4-nitro-o-phenylenediamine & 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar; preincubation

DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS: Each trial consisted of triplicate plates.

DETERMINATION OF CYTOTOXICITY
- Method: no data, but the high dose concentration was limited by toxicity.

OTHER EXAMINATIONS:
no other examinations performed
Evaluation criteria:
In this assay, a positive response is defined as a reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination.
An equivocal response is defined as an increase in revertants that is not dose related, is not reproducible, or is not of sufficient magnitude to support a determination of mutagenicity.
A negative response is obtained when no increase in revertant colonies is observed following chemical treatment.
There is no minimum percentage or fold increase required for a chemical to be judged positive or weakly positive.
Statistics:
not mandatory for this test system
Species / strain:
S. typhimurium, other: TA97, TA98, TA100, TA102 and TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
not specified
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
not specified
Additional information on results:
No details are reported.
Conclusions:
Under the test conditions reported the test substance was determined to be negative mutagenic in strains TA97, TA98, TA100, TA102 and TA1535 with and without S9.
Executive summary:

The genetic toxicity of vanadium pentoxide was assessed by testing the ability of the chemical to induce mutations in various strains of Salmonella typhimurium. Vanadium pentoxide was not mutagenic in Salmonella typhimurium strain TA97, TA98, TA100, TA102, or TA1535, with or without induced rat or hamster liver S9 enzymes.

Reason / purpose:
reference to same study
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
no data available
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Version / remarks:
1997-07-21
Deviations:
not specified
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Mice were obtained from Taconic Farms, Inc. (Germantown, NY).
- Age at study initiation: 6 or 7 weeks old
- Weight at study initiation: range of mean body weights in the exposure groups: 25-26 g (males) and 20-21 g (females)
- Assigned to test groups randomly: yes
- Housing: individually
- Diet: ad libitum; except during exposure periods
- Water: ad libitum
- Acclimation period: 10 or 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 23.9 +/- ca. 2
- Humidity (%): 55 +/- 15
- Air changes: 15/hour
- Photoperiod: 12 hours dark/light cycle
Route of administration:
inhalation: aerosol
Vehicle:
- Vehicle(s)/solvent(s) used: air
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- For the 3-month studies, vanadium pentoxide aerosol generation was based on the principle of pneumatic dispersion and consisted of two major components: a screw feeder (Model 310, Accurate, White Water, WI) that metered vanadium pentoxide powder at a constant rate and a Jet-O-Mizer jetmill (Fluid Energy Corp., Harfield, PA) that used compressed air to disperse the metered powder and form the aerosol.
- Aerosol leaving the jetmill passed through a one-stage impactor and a vertical elutriator to eliminate or deagglomerate the large particles before entering a plenum and manifold distribution system. The aerosol delivery system consisted of three holding chambers that diluted the aerosol in three stages. A metered amount of diluted aerosol was removed and mixed with conditioned air at the inlet to each exposure chamber to achieve the appropriate exposure concentration. The electrical charge buildup on the aerosol particles was neutralised by mixing the aerosol with high concentrations of bipolar ions, which were generated using a Pulse Gun (Static Control Services, Palm Springs, CA) air nozzle. A transvector air pump was installed at the aerosol inlet to each exposure chamber to provide additional control of the aerosol flow rate and improve stability of the chamber concentration.
- The stainless-steel inhalation exposure chambers (Lab Products, Inc., Maywood NJ), were designed so that uniform aerosol concentrations could be maintained throughout the chambers when catch pans were in place. The total active mixing volume of each chamber was 1.7 m³.

CHAMBER ATMOSPHERE CHARACTERISATION
- The particle size distribution in each chamber was determined prior to the start of all studies, during the first week and monthly thereafter.
- A 10-stage Quartz Crystal Microbalance-based cascade impactor was used to separate the aerosol particles into sequential size ranges; the mass median aerodynamic diameter was calculated from the corresponding mass fraction of particles at each stage.

OTHER
- The uniformity of aerosol concentration in the inhalation exposure chambers without animals was evaluated before each of the studies began; concentration uniformity with animals present in the chambers was also measured. Minor excursions in chamber uniformity values were observed in one or more exposure chambers, but these excursions had no impact on the studies.
- The stability of vanadium pentoxide in the exposure system was tested with XRD analysis. XRD analyses indicated no detectable build-up of degradation products at a detection limit of approximately 1%.
Duration of treatment / exposure:
3 months
Frequency of treatment:
6 hours plus T90 (15 minutes) per day, 5 days per week
Post exposure period:
1 day
Dose / conc.:
1 mg/m³ air (nominal)
Dose / conc.:
2 mg/m³ air (nominal)
Dose / conc.:
3 mg/m³ air (nominal)
Dose / conc.:
8 mg/m³ air (nominal)
Dose / conc.:
16 mg/m³ air (nominal)
No. of animals per sex per dose:
Groups of 10 male and 10 female mice were exposed to vanadium pentoxide.
Control animals:
yes
Positive control(s):
no data
Tissues and cell types examined:
peripheral blood samples were obtained from male and female mice
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):

DETAILS OF SLIDE PREPARATION: Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded.

METHOD OF ANALYSIS: Slides were scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) in each of nine or ten animals per exposure group.

OTHER: In addition, the ratio of polychromatic erythrocytes (PCEs) to NCEs among 1000 total erythrocytes was determined as a measure of bone marrow toxicity.
Evaluation criteria:
Statistical as well as biological factors are considered. For an individual assay, the statistical procedure for data analysis has been described in the preceding protocol. There have been instances, however, in which multiple aliquots of a chemical were tested in the same assay, and different results were obtained among aliquots and/or among laboratories. Results from more than one aliquot or from more than one laboratory are not simply combined into an overall result. Rather, all the data are critically evaluated, particularly with regard to pertinent protocol variations, in determining the weight of evidence for an overall conclusion of chemical activity in an assay.
Statistics:
The results were tabulated as the mean of the pooled results from all animals within a treatment group plus or minus the standard error of the mean. The frequency of micronucleated cells among NCEs was analyzed by a statistical software package that tested for increasing trend over exposure groups with a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposed group and the control group (ILS, 1990). In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation. In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single exposed group is less than or equal to 0.025 divided by the number of exposed groups.
Sex:
male/female
Genotoxicity:
negative
Remarks:
No increase in the frequency of micronucleated NCEs was seen in peripheral blood samples from male or female mice exposed to vanadium pentoxide for 3 months by inhalation.
Toxicity:
no effects
Remarks:
Chemical exposure had no effect on the ratio of PCEs to NCEs in peripheral blood indicating no toxicity to the bone marrow by vanadium pentoxide.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
not specified
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: Based on decreased survival in the 32 mg/m3 males and body weight decreases in 32 mg/m3 males and females, an exposure concentration of 32 mg/m3 was considered too high for use in a 3-month study. Therefore, the exposure concentrations selected for the 3-month inhalation study in rats were 0, 1, 2, 4, 8, and 16 mg/m3.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No increase in the frequency of micronucleated NCEs was seen in peripheral blood samples from male or female mice exposed to vanadium pentoxide for 3 months by inhalation.
- Ratio of PCE/NCE (for Micronucleus assay): Chemical exposure had no effect on the ratio of PCEs to NCEs in peripheral blood (data not presented), indicating no toxicity to the bone marrow by vanadium pentoxide.
Conclusions:
Vanadium pentoxide, administered for 3 months by inhalation to male and female mice, did not increase the frequency of micronucleated normochromatic erythrocytes in peripheral blood.
Executive summary:

The genetic toxicity of vanadium pentoxide was assessed by testing the ability of the chemical to induce an increases in the frequency of micronucleated erythrocytes in mouse peripheral blood. Mice were exposed 90 days to an vanadium pentaoxide aerosol by inhalation before blood was sampled and prepared for analysis.

Vanadium pentoxide, administered to male and female mice, did not increase the frequency of micronucleated normochromatic erythrocytes in peripheral blood.

Reason / purpose:
reference to same study
Reference
Endpoint:
carcinogenicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-01-06 (first exposure) to 1999-01-04/08 (necropsy date)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Male and female rats (N=50/sex/dose) were exposed to various concentrations of V2O5 for 6 hours/d, for 5 d/wk and for a period of 16 days, 3 month or 2 years. Clinical signs, body weights, blood and urine were observed during the study. Surviving animals were sacrificed at study end and necropsy was performed. Appearance of neoplasms in exposed animals compared to controls was analysed. Male and female F344 rats were exposed to 0, 0,5 and 1 mg/m3 V2O5.
GLP compliance:
yes
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: average age: 6 or 7 weeks old on the first day of the study
- Weight at study initiation: average body weight for week 1: 134-136 g (males) and 130-105 g (females)
- Housing: housed individually; stainless steel wire bottom (Hazleton Systems, Inc., Aberdeen, MD); cages and racks were rotated weekly.
- Diet: ad libitum, except during exposure periods; NTP-2000 pelleted diet (Zeigler Brothers, Inc., Gardeners, PA), changed weekly
- Water: ad libitum; tap water (Richland, WA, municipal supply water used) via automatic watering system
- Acclimation period: quarantined for 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 23.9± ca.2°C (75° ± 3° F)
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day
Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1 - <= 1.3 µm
Remarks on MMAD:
GSD = 2.3 - 2.8
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- MMAD = 1.0-1.3 µm, GSD = 2.3-2.8
- The generation and delivery system used in the 16-day special studies and the 2-year studies consisted of a linear dust feeder, a particle attrition chamber, and an aerosol distribution system. The linear dust feeder, a slide-bar dust-metering device, was composed of a shuttle bar, body, outlet port, and hopper. As the compressed-air-driven shuttle bar slid back and forth during generation, the metering port aligned with the hopper, which served as a reservoir for the bulk chemical, and was filled with a small amount of vanadium pentoxide powder. As the shuttle bar slid to the dispersing position, the metering port aligned with a compressed-air port in the body and a puff of air from this port dispersed the vanadium pentoxide into the particle attrition chamber. Generator output was regulated by adjusting the cadence of the shuttle bar. The particle attrition chamber used low fluid energy from an air jet tangential to the chamber to deagglomerate the vanadium pentoxide particles. After deagglomeration, the particles were swept into a classification zone where smaller particles exited to the distribution line; larger particles were thrown to the perimeter of the classifier by centrifugal force and were reentrained into the impacting air jet, and the process was repeated until the particles were sufficiently deagglomerated. The aerosol passed through the distribution lines to the exposure chambers. A pneumatic pump designed by the study laboratory was located at each chamber inlet and drew aerosol from the distribution line into the chamber inlet, where it was diluted with conditioned air to the appropriate concentration. Flow through the distribution line was controlled by Air-Vac pumps (Air-Vac Engineering, Milford, CT), and pressure was monitored by photohelic differential pressure gauges (Dwyer Instruments, Inc., Michigan City, IN).
- The Stainless steel chambers (Lab Products, Inc., Harford Systems Division, Aberdeen, MD), were designed so that uniform aerosol concentrations could be maintained throughout the chambers when catch pans were in place. The total active mixing volume of each chamber was 1.7 m³.

CHAMBER ATMOSPHERE CHARACTERIZATION
- The particle size distribution in each chamber was determined prior to the start of all studies, during the first week of the 16-day and 3-month studies, during the first 2 weeks of the 2-year studies, and monthly during the 3-month and 2-year studies.
- For the 16-day special studies and the 2-year studies, a Mercer-style seven-stage impactor was used. The stages (glass coverslips lightly sprayed with silicon) were analyzed by ICP/AES, and the relative mass collected on each stage was analyzed by probit analysis.

OTHER
- The uniformity of aerosol concentration in the inhalation exposure chambers without animals was evaluated before each of the studies began; concentration uniformity with animals present in the chambers was also measured. During the 16-day and 3-month studies, minor excursions in chamber uniformity values were observed in one or more exposure chambers, but these excursions had no impact on the studies. Chamber concentration uniformity was acceptable throughout the 16-day special studies and 2-year studies.
- The stability of vanadium pentoxide in the exposure system was tested with XRD analysis. XRD analyses indicated no detectable build-up of degradation products at a detection limit of approximately 1%.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- During all studies, chamber aerosol concentrations were monitored with real-time aerosol monitors (RAMs) that used a pulsed-light-emitting diode in combination with a silicon detector to sense light scattered over a forward angular range of 45° to 95° by particles traversing the sensing volume. The instruments respond to particles 0.1 to 20 μm in diameter.
- For the 16-day special studies and the 2-year studies, the sampling system consisted of a valve that multiplexed each RAM to two or three exposure chambers and to a HEPA filter and/or the control chamber or room; selection of sampling streams and data acquisition from each RAM was remotely controlled by a computer. Equations for calibration curves were stored in the computers and were used to convert the measured voltages to exposure concentrations.
- Each RAM was calibrated daily during the 16-day and 3-month studies by correlating the measured voltage with vanadium pentoxide concentrations determined by gravimetric analysis of glass fiber filters and one to two times per week during the 2-year studies by ICP/AES or ICP/mass spectrometry analysis of Pallflex® TX40H120WW glass fiber filters.
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
6 hours per day, 5 days per week
Post exposure period:
no
Dose / conc.:
0.5 mg/m³ air (nominal)
Dose / conc.:
1 mg/m³ air (nominal)
Dose / conc.:
2 mg/m³ air (nominal)
No. of animals per sex per dose:
core study: 50 male and 50 female rats
tissue burden analyses: 40 female rats per exposed group; separate control group of 15 female rats was used as chamber controls
Control animals:
yes
Details on study design:
- Dose selection rationale: based on the incidences and severities of respiratory lesions and increased lung weights in male and female rats in the 90-day study, concentrations of 4 mg/m3 or greater were considered to be too high for use in a 2-year study. The exposure concentrations selected for the 2-year inhalation study in rats were 0.5, 1, and 2 mg/m3.
- Rationale for animal assignment (if not random): randomly into groups of approximately equal initial mean body weights.
Positive control:
not stated
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Clinical findings were recorded every 4 weeks from week 5 through 89 and every 2 weeks from week 92 until the end of the studies
- The health of the animals was monitored during the studies according to the protocols of the NTP Sentinel Animal Program

BODY WEIGHT: Yes
- Time schedule for examinations: body weights were recorded on day 1 and body weights were recorded every 4 weeks from week 5 through 89 and every 2 weeks from week 92 until the end of the studies

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

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 data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
- Tissue Burden Studies: groups of five female rats were evaluated on days 1, 5, 12, 26, 54, 173, 360, and 542; total lung weight, right lung burden, and left lung histopathology were measured in exposed animals at all time points.
- Blood vanadium concentration was measured in all animals at all time points after day 12. Groups of five chamber control animals were bled at each of these time points and returned to their chambers and used for subsequent bleedings. Blood was obtained by cardiac puncture from exposed animals or from the retroorbital sinus of chamber control animals.
Sacrifice and pathology:
Method of sacrifice: CO2 asphyxiation.

PATHOLOGY: Yes
- Necropsy was performed at study end on all core study animals.
- All organs and tissues were examined for grossly visible lesions, and all major tissues were prepared for microscopic examination.

HISTOPATHOLOGY: Yes
- Complete histopathology was performed on all core study animals. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gallbladder (mice only), heart and aorta, large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum, and ileum), kidney, larynx, liver, lung and mainstem bronchi, lymph nodes (mandibular, mediastinal, mesenteric, and bronchial), mammary gland (except male mice), nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder and uterus.
Other examinations:
Five male and five female rats were randomly selected for parasite evaluation and gross observation of disease.
Statistics:
Survival Analyses:
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. Animals found dead of other than natural causes were censored from the survival analyses; animals dying from natural causes were not censored. Statistical analyses for possible doserelated effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.

Analysis of Continuous Variables:
Two approaches were employed to assess the significance of pairwise comparisons between exposed and control groups in the analysis of continuous variables. Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Hematology, clinical chemistry, urinalysis, urine concentrating ability, cardiopulmonary, immunotoxicologic, cell proliferation, tissue concentrations, spermatid, and epididymal spermatozoal data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test). Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973). Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across exposure concentrations. (for more information see publication)
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
only marginally less for the 2 mg/m3–exposed females
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Non-neoplastic lesions occurred in respiratory system of males and females (lung, larynx, and nose) , and the severities of these lesions generally increased with increasing exposure concentration.

LUNGS:
- Effects in males: alveolar epithelium, hyperplasia (7/50, 24/49, 34/48, 49/50); bronchiole, epithelium hyperplasia (3/50, 17/49, 31/48, 49/50); alveolar epithelium, metaplasia, squamous (1/50, 0/49, 0/48, 21/50); bronchiole, metaplasia, squamous (0/50, 0/49, 0/48, 7/50); inflammation, chronic active (5/50, 8/49, 24/48, 42/50); interstitial, fibrosis (7/50, 7/49, 16/48, 38/50); alveolus, infiltration cellular, histiocyte (22/50, 40/49,45/48, 50/50).
- Effects in females: alveolar epithelium, hyperplasia (4/49, 8/49, 21/50, 50/50); bronchiole, epithelium hyperplasia (6/49, 5/49, 14/50, 48/50); alveolar epithelium, metaplasia, squamous (0/49, 0/49, 0/50, 6/50); inflammation, chronic active (10/49, 10/49, 14/50, 40/50); interstitial, fibrosis (19/49, 7/49, 12/50, 32/50); alveolus, infiltration cellular, histiocyte (26/49, 35/49, 44/50, 50/50).
- Incidences of minimal to mild chronic active inflammation and interstitial fibrosis in the lungs were significantly increased in males exposed to 1 or 2 mg/m3 and females exposed to 2 mg/m3, and the incidences of histiocytic cellular infiltrate of the alveolus were increased in all exposed groups of males and females. The inflammatory lesions were primarily minimal to mild and consisted of interstitial and perivascular infiltrates of mostly mononuclear inflammatory cells that were occasionally within alveoli. Alveolar septa were occasionally thickened by thin strands of eosinophilic fibrillar material (fibrosis). The histiocytic infiltrate was also minimal to mild, consisting of scattered intraalveolar macrophages that contained large amounts of foamy intracytoplasmic material, interpreted as pulmonary surfactant. Additionally, scant amounts of eosinophilic material (surfactant) similar to that observed within alveolar macrophages was also free within alveoli; however, a separate diagnosis was not made. A brownish pigment (pigmentation) was visible in alveolar macrophages in some males and females exposed to 2 mg/m3 and in females exposed to 1 mg/m3; it was a mild change considered of little biological significance and was not further characterized.

LARYNX:
- Effects in males: inflammation, chronic (3/49, 20/50, 17/50, 28/49); respiratory epithelium, epiglottis degeneration (0/49, 22/50, 23/50, 33/49); respiratory epithelium, epiglottis, hyperplasia (0/49, 18/50, 34/50, 32/49); respiratory epithelium, epiglottis, metaplasia, squamous (0/49, 9/50, 16/50, 19/49).
- Effects in females: inflammation, chronic (8/50, 26/49, 27/49, 37/50); respiratory epithelium, epiglottis degeneration (2/50, 33/49, 26/49, 40/50);
respiratory epithelium, epiglottis, hyperplasia (0/50, 25/49, 26/49, 33/50); respiratory epithelium, epiglottis, metaplasia, squamous (2/50, 7/49, 7/49, 16/50).
- There were increased incidences of minimal to mild lesions of the larynx in exposed males and females. The incidences generally increased with increasing exposure concentration and included chronic inflammation of the larynx and degeneration, hyperplasia, and squamous metaplasia of the respiratory epithelium of the epiglottis. The inflammation consisted of a mixture of mononuclear and granulocytic inflammatory cells in the submucosa beneath the epithelium lining the base of the epiglottis, ventral pouch, and caudal larynx. The degeneration of the respiratory epithelium was characterized by a loss or decrease in the height of cilia and shortening of the normally columnar to cuboidal surface epithelial cells lining the laryngeal surface of the base of the epiglottis. Squamous metaplasia was diagnosed when the ciliated cells were replaced by one or more layers of flattened squamous epithelium. In the same area, the respiratory epithelium was mildly thickened in many animals; this change was diagnosed as hyperplasia. These changes are relatively minimal, commonly occur in rats in NTP inhalation studies, and represent a common response to laryngeal injury.

NOSE:
- Effects in males: goblet cell, respiratory epithelium, hyperplasia (4/49, 15/50, 12/49, 17/48)
- Effects in females: goblet cell, respiratory epithelium, hyperplasia (13/50, 18/50, 16/50, 30/50)
- There were increased incidences of mild goblet cell hyperplasia of the nasal respiratory epithelium in all groups of exposed male rats and in females exposed to 2 mg/m3. Increased numbers of goblet cells were most notable in the respiratory epithelium lining the median septum adjacent to the area of the vomeronasal organ.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Based on the analysis of Starr et al. (2012), effects oberved in the male rat are not significant.
Other effects:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
- Survival of the animals were similar to the controls

BODY WEIGHT AND WEIGHT GAIN
- Body weights of the animals were similar to the controls except body weight of the 2 mg/m3–exposed females which was less

HISTOPATHOLOGY: NON-NEOPLASTIC
KIDNEY:
- The incidences of nephropathy were significantly increased in male rats exposed to 1 or 2 mg/m3. Nephropathy is a common lesion in aged rats, particularly males, and has been diagnosed in virtually all males in NTP 2-year studies that used the NIH-07 diet. In those studies, chemical exacerbation of nephropathy was identified by increased severity. With the NTP-2000 diet, the severity of spontaneous nephropathy has been reduced. In this study, the severity of nephropathy was not increased in exposed groups of males. Also, exposed females were not affected. Although the NTP doesn’t have a formal historical control database for nonneoplastic lesions, a review of recent studies indicates that the incidence in the male chamber control group in the current study is low. It is not clear if the increased incidences in this study were related to exposure to vanadium or were a reflection of the low incidence in the control group. Regardless, nephropathy was a relatively weak response and was likely of marginal biological significance.

HISTOPATHOLOGY: NEOPLASTIC (if applicable):

Please note that the following carcinogenic effects as reported in the original study are not statistically significant according to Starr et al. (2012).

LUNG:
- Effects in males: alveolar/ bronchiolar adenoma (4/50, 8/49, 5/48, 6/50); alveolar/bronchiolar carcinoma (0/50, 3/49, 1/48, 3/50); alveolar/ bronchiolar adenoma or carcinoma (4/50, 10/49, 6/48, 9/50)
- Effects in females: none (equivocal findings: alveolar/bronchiolar adenoma (0/49, 3/49, 1/50, 0/50); alveolar/bronchiolar adenoma or carcinoma (0/49, 3/49, 1/50, 1/50)
- Alveolar/bronchiolar neoplasms were present in exposed groups of males and one 2 mg/m3 female. Alveolar/bronchiolar adenomas were present in 0.5 and 1 mg/m3 females. Additionally, one female exposed to 2 mg/m3 had an alveolar/bronchiolar carcinoma. There were no statistically significant increases in the incidences of lung neoplasms in rats.
- There were increased incidences of alveolar epithelial hyperplasia and bronchiole hyperplasia in the lungs of males exposed to 0.5 mg/m3 or greater and females exposed to 1 or 2 mg/m3. The severities of these lesions were increased in 2 mg/m3 males and females. In affected animals, this was essentially a diffuse change with proliferation of epithelium in the distal terminal bronchioles and immediately associated alveolar ducts and alveoli. Normally flattened epithelium was replaced with cuboidal epithelium.
- Increased incidences of squamous metaplasia of the alveoli occurred in male and, to a lesser extent, in female rats exposed to 2 mg/m3. There were a spectrum of changes ranging from minimal to severe. Minimal lesions were characterized by a single alveolus with the thin type I cells which normally line alveoli replaced by one to several layers of squamous epithelium. Severe lesions were much larger, often involving an area approximately 1 cm in diameter. Many alveoli were involved and there was apparent coalescence of the metaplasia. There were also lesions of intermediate severity. Keratin production was a prominent feature of the squamous metaplasia observed in this study. Keratin often filled the affected alveoli, and in some of the lesions, cyst-like structures filled with keratinous material were formed. In a few animals (predominantly males), the squamous metaplasia extended into the distal airways and was diagnosed as bronchiole squamous metaplasia. Commonly dispersed within the squamous lesions were areas of respiratory epithelial metaplasia in which the alveolar epithelium was replaced by tall cuboidal to columnar epithelium with cilia often present and with mucous material filling the alveolar lumen.

UTERUS:
- The incidences of stromal polyp occurred with a positive trend in female rats (chamber control, 6/50; 0.5 mg/m3, 3/50; 1 mg/m3, 7/50; 2 mg/m3, 13/50). However, the incidence in the 2 mg/m3 group was within the historical range in controls. Endometrial stromal polyps are common neoplasms in the F344/N rat in NTP studies. They are benign neoplasms and generally do not progress to malignancy; however, they occasionally do progress to stromal sarcoma. In this study, when the incidences of stromal polyp were combined with the single incidence of stromal sarcoma, the combined incidence in 2 mg/m3 females was significantly increased. The marginal increase in the incidence of stromal polyp and stromal sarcoma (combined) in females exposed to 2 mg/m3 was not considered related to exposure to vanadium pentoxide.

OTHER FINDINGS
LUNG BURDEN STUDIES:
- Histopathology: the left lung lobe from each animal was infused with 10% neutral buffered formalin, and sections were examined microscopically. The purpose was to follow progression of the lung lesions. Following day 1 of exposure, there was an infiltrate of alveolar macrophages in the lungs. With continued exposure, increased numbers of alveolar macrophages, interstitial mononuclear inflammatory cell infiltrates, and hyperplasia of alveolar and bronchiolar epithelium were observed. In rats exposed to 2 mg/m3, there was an increase in severity of the hyperplasia between days 54 and 173. An increase in severity was not obvious between days 173 and 360, but hyperplasia appeared more severe on day 542. Hyperplasia was observed in only a few animals exposed to 1 mg/m3 and only on day 542. The minimal fibrosis observed in the 2-year study was not readily apparent on day 542 or earlier.
- Lung weights from exposed female rats increased throughout the study. Although there appeared to be an exposure concentration-related increase in lung weights after day 26 of the study, it was primarily due to increases in lung weights of female rats exposed to 2 mg/m3. In general, lung weights of 0.5 or 1 mg/m3 females were similar.
- Lung burden data appeared proportional to exposure concentration in rats.
- Though deposition patterns were similar between rats and mice, the maximum lung burdens occurred at day 173 in rats. The lung burdens appeared to reach steady state at the lowest exposure concentrations (0.5 mg/m3). A decline in lung burdens was observed. The retention of vanadium in the lungs at 18 months was ca. 13% to 15% in rats. The total lung doses for rats exposed to 0.5, 1, or 2 mg/m3 were estimated to be 130, 175, and 308 μg vanadium, respectively.
- Lung clearance half-times were considerably longer than those observed in the 16-day special studies.

BLOOD:
- Vanadium was detected in the blood at concentrations several orders of magnitude lower than those measured in the lungs of exposed rats, and blood vanadium concentrations in exposed groups were only marginally increased over that of the chamber control group. Overall, blood vanadium concentrations appeared to increase with increasing exposure concentration; however, this proportionality was less clear when the 0.5 and 1 mg/m3 groups were compared.
- Blood vanadium concentrations in all exposed groups appeared to peak on days 26 or 54 after which there was a decline throughout the rest of the study. This response was similar to that seen in lung burdens. However, these changes in concentrations were small, making it difficult to determine if there was an increase in elimination of vanadium from the blood or a decreased absorption from the lung due to reduced deposition, especially at the higher exposure concentrations.
Relevance of carcinogenic effects / potential:
In the 2-year study, some evidence of carcinogenic activity for male rats and equivocal evidence for carcingenic activity for female rats was reported. Based on the analysis of Starr et al. (2012), the observed carconogenic effects are statistically not significant as follows:
(1) there are not any statistically significant differences in tumor incidence between vanadium pentoxide-treated and concurrent control group male and female rats,
(2) there is weakened evidence from comparisons with the widened historical control tumor incidence ranges that result from use of updated historical control data, and
(3) there is a likelihood that all of the male rats utilized in the vanadium pentoxide bioassay may have had elevated risks of developing alveolar/bronchiolar adenoma even in the absence of vanadium pentoxide exposure.
The genetic toxicology studies ( Salmonella typhimurium gene mutations and Micronucleated erythrocytes Mouse peripheral blood in vivo) show negative results for mutagenic effects.
Dose descriptor:
LOAEC
Effect level:
0.5 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Chamber concentration uniformity was acceptable throughout the 16-day special studies and 2-year studies.

Conclusions:
Survival rates and body weights were not affected in rats exposed to vanadium pentoxide for 2 years. As in the 3-month studies, the respiratory tract was the primary site of toxicity in rats. Exposure to vanadium pentoxide caused a spectrum of nonneoplastic 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 rats and an unusual squamous metaplasia of the lung in male and female rats.
Reason / purpose:
reference to same study
Reference
Endpoint:
carcinogenicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1997-01-13 (first exposure) to 1999-01-11/15 (necropsy date)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well reported study. Test procedure in accordance with generally accepted scientific standards and described in sufficient detail.
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Male and female mice (N=50/sex/dose) were exposed to various concentrations of V2O5 for 6 hours/d, for 5 d/wk and for a period of 16 days, 3 month or 2 years. Clinical signs, body weights, blood and urine were observed during the study. Surviving animals were sacrificed at study end and necropsy was performed. Appearance of neoplasms in exposed animals compared to controls was analysed. Male and female B6C3F1 mice were exposed to 0, 1, 2 and 4 mg/m3 V2O5.
GLP compliance:
yes
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: average age: 6 or 7 weeks old on the first day of the study
- Weight at study initiation: average body weight for week 1: 24.0-24.4 g (males) and 19.6-19.9 g (females)
- Housing: housed individually; stainless steel wire bottom (Hazleton Systems, Inc., Aberdeen, MD); cages and racks were rotated weekly.
- Diet: ad libitum, except during exposure periods; NTP-2000 pelleted diet (Zeigler Brothers, Inc., Gardeners, PA), changed weekly
- Water: ad libitum; tap water (Richland, WA, municipal supply water used) via automatic watering system
- Acclimation period: quarantined for 16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 23.9± ca.2°C (75° ± 3° F)
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day

IN-LIFE DATES: From: 1997-01-13 (first exposure) to 1999-01-11/15 (necropsy date)
- No further information on test material was stated.
Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- MMAD = 1.0-1.3 µm, GSD = 2.3-2.8
- The generation and delivery system used in the 16-day special studies and the 2-year studies consisted of a linear dust feeder, a particle attrition chamber, and an aerosol distribution system. The linear dust feeder, a slide-bar dust-metering device, was composed of a shuttle bar, body, outlet port, and hopper. As the compressed-air-driven shuttle bar slid back and forth during generation, the metering port aligned with the hopper, which served as a reservoir for the bulk chemical, and was filled with a small amount of vanadium pentoxide powder. As the shuttle bar slid to the dispersing position, the metering port aligned with a compressed-air port in the body and a puff of air from this port dispersed the vanadium pentoxide into the particle attrition chamber. Generator output was regulated by adjusting the cadence of the shuttle bar. The particle attrition chamber used low fluid energy from an air jet tangential to the chamber to deagglomerate the vanadium pentoxide particles. After deagglomeration, the particles were swept into a classification zone where smaller particles exited to the distribution line; larger particles were thrown to the perimeter of the classifier by centrifugal force and were reentrained into the impacting air jet, and the process was repeated until the particles were sufficiently deagglomerated. The aerosol passed through the distribution lines to the exposure chambers. A pneumatic pump designed by the study laboratory was located at each chamber inlet and drew aerosol from the distribution line into the chamber inlet, where it was diluted with conditioned air to the appropriate concentration. Flow through the distribution line was controlled by Air-Vac pumps (Air-Vac Engineering, Milford, CT), and pressure was monitored by photohelic differential pressure gauges (Dwyer Instruments, Inc., Michigan City, IN).
- The Stainless steel chambers (Lab Products, Inc., Harford Systems Division, Aberdeen, MD), were designed so that uniform aerosol concentrations could be maintained throughout the chambers when catch pans were in place. The total active mixing volume of each chamber was 1.7 m³.

CHAMBER ATMOSPHERE CHARACTERIZATION
- The particle size distribution in each chamber was determined prior to the start of all studies, during the first week of the 16-day and 3-month studies, during the first 2 weeks of the 2-year studies, and monthly during the 3-month and 2-year studies.
- For the 16-day special studies and the 2-year studies, a Mercer-style seven-stage impactor was used. The stages (glass coverslips lightly sprayed with silicon) were analyzed by ICP/AES, and the relative mass collected on each stage was analyzed by probit analysis.

OTHER
- The uniformity of aerosol concentration in the inhalation exposure chambers without animals was evaluated before each of the studies began; concentration uniformity with animals present in the chambers was also measured. During the 16-day and 3-month studies, minor excursions in chamber uniformity values were observed in one or more exposure chambers, but these excursions had no impact on the studies. Chamber concentration uniformity was acceptable throughout the 16-day special studies and 2-year studies.
- The stability of vanadium pentoxide in the exposure system was tested with XRD analysis. XRD analyses indicated no detectable build-up of degradation products at a detection limit of approximately 1%.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- During all studies, chamber aerosol concentrations were monitored with real-time aerosol monitors (RAMs) that used a pulsed-light-emitting diode in combination with a silicon detector to sense light scattered over a forward angular range of 45° to 95° by particles traversing the sensing volume. The instruments respond to particles 0.1 to 20 μm in diameter.
- For the 16-day special studies and the 2-year studies, the sampling system consisted of a valve that multiplexed each RAM to two or three exposure chambers and to a HEPA filter and/or the control chamber or room; selection of sampling streams and data acquisition from each RAM was remotely controlled by a computer. Equations for calibration curves were stored in the computers and were used to convert the measured voltages to exposure concentrations.
- Each RAM was calibrated daily during the 16-day and 3-month studies by correlating the measured voltage with vanadium pentoxide concentrations determined by gravimetric analysis of glass fiber filters and one to two times per week during the 2-year studies by ICP/AES or ICP/mass spectrometry analysis of Pallflex® TX40H120WW glass fiber filters.
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
6 hours per day, 5 days per week
Remarks:
Doses / Concentrations:
1 mg/m³ V2O5
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2 mg/m³ V2O5
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
4 mg/m³ V2O5
Basis:
nominal conc.
No. of animals per sex per dose:
core study: 50 males and 50 females per dose
tissue burden analyses: 40 female mice per exposed group; separate control group of 15 female mice was used
Control animals:
yes
Details on study design:
- Dose selection rationale: the highest exposure concentration (4 mg/m3) selected for mice was based on reduced body weight gains of 8 and 16 mg/m3 males and females, increased lung weights of 4 mg/m3 or greater males and females, increased incidences of epithelial hyperplasia, and increased incidences and severities of inflammatory lesions of the lungs in mice exposed to 8 mg/m3 versus those exposed to 4 mg/m3 in the 90-day study.
- Rationale for animal assignment (if not random): randomly into groups of approximately equal initial mean body weights.
- No further information on test material was stated.
Positive control:
not stated
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Clinical findings were recorded every 4 weeks from week 5 through 89 and every 2 weeks from week 92 to the end of the study.

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed at the beginning of the studies. Body weights were recorded every 4 weeks from week 5 through 89 and every 2 weeks from week 92 to the end of the study.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

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 data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
Tissue Burden Studies:
- Lungs and whole blood were collected from five mice in each exposure group sacrificed on day 1, 5, 12, 26, 54, 171, 362, or 535. Groups of five chamber control animals were bled at each of these time points and returned to their chambers and used for subsequent bleedings. The lungs and associated tissues were removed from exposed animals; after the left and right lung were prepared for histopathologic examination, and the right lung was digested to measure vanadium concentration. Vanadium concentrations in blood and right lung nitric acid digests were measured. Calculations of lung deposition and clearance parameters were performed. Blood vanadium concentration was measured in all animals at all time points after day 12.
Sacrifice and pathology:
Method of sacrifice: CO2 asphyxiation.

PATHOLOGY: Yes
- Complete necropsies were performed on all core study mice.
- All organs and tissues were examined for grossly visible lesions, and all major tissues were prepared for microscopic examination.

HISTOPATHOLOGY: Yes.
- Complete histopathology was performed on all core study animals. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gallbladder (mice only), heart and aorta, large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum, and ileum), kidney, larynx, liver, lung and mainstem bronchi, lymph nodes (mandibular, mediastinal, mesenteric, and bronchial), mammary gland (except male mice), nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder and uterus.
Other examinations:
Five male and five female mice were randomly selected for parasite evaluation and gross observation of disease.
Statistics:
Survival Analyses:
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. Animals found dead of other than natural causes were censored from the survival analyses; animals dying from natural causes were not censored. Statistical analyses for possible doserelated effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.

Analysis of Continuous Variables:
Two approaches were employed to assess the significance of pairwise comparisons between exposed and control groups in the analysis of continuous variables. Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Hematology, clinical chemistry, urinalysis, urine concentrating ability, cardiopulmonary, immunotoxicologic, cell proliferation, tissue concentrations, spermatid, and epididymal spermatozoal data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test). Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973). Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across exposure concentrations. (for more information see publication)
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY
- Survival of the highest dosed males (4 mg/m³) was significantly less than controls
- Abnormal breathing was observed particularly those exposed to 2 or 4 mg/m³
- Many mice exposed to vanadium pentoxide were thin

BODY WEIGHT AND WEIGHT GAIN
-Mean body weight of all females and highest dosed males was below the controls; and mean body weights of males exposed to 2 mg/m3 were less from week 85 to the end of the study

HISTOPATHOLOGY: NON-NEOPLASTIC
- Non-neoplastic lesions occurred in respiratory system (lung, larynx, and nose) of males and females, and the severities of these lesions generally increased with increasing exposure concentration.
-Bronchial lymph node hyperplasia was present in most of the exposed females.

LUNG:
- There were significantly increased incidences of alveolar epithelial hyperplasia and bronchiolar epithelial hyperplasia in the lungs of exposed male and female mice. Incidences of chronic inflammation and histiocytic cellular infiltrate were significantly increased in exposed groups of mice, and the incidences of interstitial fibrosis were increased in mice exposed to 2 or 4 mg/m3.
- Males: alveolar epithelium, hyperplasia (3/50, 41/50, 49/50, 50/50); bronchiole, epithelium, hyperplasia (0/50, 15/50, 37/50, 46/50); inflammation chronic (6/50, 42/50, 45/50, 47/50); alveolus, infiltration cellular, histiocyte (10/50, 36/50, 45/50, 49/50); interstitial fibrosis (1/50, 6/50, 9/50, 12/50)
- Females: alveolar epithelium, hyperplasia (0/50, 31/50, 38/50, 50/50); bronchiole, epithelium, hyperplasia (0/50, 12/50, 34/50, 48/50); inflammation chronic (4/50, 37/50, 39/50, 49/50); alveolus, infiltration cellular, histiocyte (0/50, 34/50, 35/50, 45/50); interstitial fibrosis (0/50, 1/50, 4/50,
8/50)
- The hyperplasia was essentially a diffuse change with proliferation of epithelium in the distal terminal bronchioles and immediately associated alveolar ducts and alveoli. Normally flattened epithelium was replaced with cuboidal epithelium. The hyperplasia of the alveolar epithelium was pronounced and increased in severity with increasing exposure concentration, while the hyperplasia of the distal bronchioles was minimal to mild with slight increases in severity in mice exposed to 4 mg/m3. The changes in mice were similar to those observed in rats but were not as pronounced.
- The inflammatory lesions were primarily minimal to mild and consisted of interstitial, perivascular, and peribronchiolar infiltrates of mostly mononuclear inflammatory cells (mostly lymphocytes) that were occasionally present within alveoli. The most prominent histiocytic infiltrate occurred within alveoli in close proximity to alveolar/bronchiolar neoplasms, particularly carcinomas.

LARYNX:
- There were significantly increased incidences of minimal squamous metaplasia of the respiratory epithelium of the epiglottis in exposed groups.
- Males: respiratory epithelium, epiglottis, metaplasia, squamous (2/49, 45/50, 41/48, 41/50)
- Females: respiratory epithelium, epiglottis,metaplasia, squamous (0/50, 39/50, 45/49, 44/50)
- Squamous metaplasia was diagnosed when the ciliated cells were replaced by one to three layers of flattened squamous epithelium. This change commonly occurs in mice in NTP inhalation studies and represents a common response to laryngeal injury.

NOSE:
- There were increased incidences of minimal to mild suppurative inflammation of the nose in males and females exposed to 2 or 4 mg/m3.
- Males: inflammation suppurative (16/50, 11/50, 32/50, 23/50); olfactory epithelium, degeneration, hyaline (1/50, 7/50, 23/50, 30/50); respiratoryepithelium, degeneration, hyaline (8/50, 22/50, 38/50, 41/50); respiratory epithelium, metaplasia, squamous (0/50, 6/50, 6/50, 2/50)
- Females: inflammation suppurative (19/50, 14/50, 32/50, 30/50); olfactory epithelium, atrophy (2/50, 8/50, 5/50, 14/50); olfactory epithelium, degeneration, hyaline (11/50, 23/50, 34/50, 48/50); respiratory epithelium, degeneration, hyaline (35/50, 39/50, 46/50, 50/50); respiratory epithelium, metaplasia, squamous (0/50, 3/50, 7/50, 8/50); respiratory epithelium, necrosis (0/50, 0/50, 1/50, 7/50)
- The inflammation consisted of focal aggregates of few to moderate numbers of neutrophils generally subjacent to the epithelium of the turbinates, septum, or lateral wall of the anterior nose. In the more severe cases (predominantly in females exposed to 4 mg/m3), a short segment of the overlying epithelium was ulcerated (necrosis). Similarly, in some males and females, the overlying respiratory epithelium was replaced by one or more layers of flattened epithelium (squamous metaplasia), and the incidences were marginally increased in some exposed groups of mice.
- The majority of the olfactory epithelium covers the turbinates in the distal portion of the nose. There were marginal but significant increases in the incidences of atrophy of this epithelium in females exposed to 1 or 4 mg/m3.

BRONCIAL LYMPH NODE
- There were significant increases in the incidences of hyperplasia of the bronchial lymph node in exposed groups of females, and while not significant, a positive trend in the incidences of this lesion also occurred in males.
- Females: hyperplasia (3/39, 13/40, 14/45, 20/41)

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
LUNG:
- The incidences of alveolar/bronchiolar carcinoma and alveolar/bronchiolar adenoma or carcinoma (combined) were significantly increased in all groups of exposed male and female mice compared to controls
- Males: alveolar/bronchiolar adenoma (13/50, 16/50, 26/50, 15/50); alveolar/ bronchiolar carcinoma (12/50, 29/50, 30/50, 35/50); alveolar/ bronchiolar adenoma or carcinoma (22/50, 42/50, 43/50, 43/50)
- Females: alveolar/bronchiolar adenoma (1/50, 17/50, 23/50, 19/50); alveolar/ bronchiolar carcinoma (0/50, 23/50, 18/50, 22/50); alveolar/ bronchiolar adenoma or carcinoma (1/50, 32/50, 35/50, 32/50)
- The incidences of alveolar/bronchiolar adenoma were significantly increased in males exposed to 2 mg/m3 and in all groups of exposed females.
- These increased incidences exceeded the historical ranges for controls.

SPLEEN:
- There was a positive trend in the incidences of hemangiosarcoma of the spleen in male mice (chamber control, 0/50; 1 mg/m3, 0/50; 2 mg/m3, 0/50; 4 mg/m3, 3/50).
- However, the incidence in the 4 mg/m3 group was within the historical range in controls.
- In addition, the incidences of hemangioma or hemangiosarcoma (combined) in all organs were not significant. The marginal increase in the incidence of hemangiosarcoma of the spleen was not considered related to vanadium pentoxide exposure.

OTHER FINDINGS
TISSUE BURDEN STUDIES:
- Histopathology: lung lesions were identified early, and were more severe in females exposed to higher concentrations and progressed with time. Minimal bronchiolar epithelial hyperplasia and interstitial inflammation were initially observed in the 4 mg/m3 group on day 5. Bronchiolar epithelial hyperplasia was characterized by slight piling up of cells, some nuclear pleomorphism, and occasional karyorrhectic cells. On day 12, exposure-related lesions were evident in 2 mg/m3 females, while alveolar epithelial hyperplasia was more prominent in the 4 mg/m3 group. Lesions were first observed in the 1 mg/m3 group on day 54, and lesion severities in the 2 and 4 mg/m3 groups appeared to be increased on day 54. On day 362, lesion severity in lungs of females exposed to 4 mg/m3 was noticeably increased, and bronchiolar hyperplasia included more nuclear pleomorphism. On day 535, mice exposed to 2 mg/m3 had hyperplastic alveolar epithelial lesions and, similar to females exposed to 4 mg/m3, the hyperplasia was oriented around the terminal bronchiolar/alveolar duct. Also on day 535, lung nodules/masses were observed. While some were confirmed as neoplasms, many were in the right lung lobe and thus were not confirmed histopathologically. Similar lesions, although more severe, were observed in the core group of animals at 2 years.
- Lung burden data appeared proportional to exposure concentration in mice. Rats removed vanadium from the lungs much slower than mice, with clearance half-times of vanadium lung burdens approximately six to nine fold longer in rats than in mice at comparable exposure concentrations. In rats and mice, lung clearance half-times were considerably longer than those observed in the 16-day special studies.
- The maximum lung burdens occurred between days 26-54 in mice. The lung burdens appeared to reach steady state at the lowest exposure concentrations (1 mg/m3). A decline in lung burdens was observed. The retention of vanadium in the lungs at 18 months was 2% to 3% in mice. The total lung doses for mice exposed to 1, 2, or 4 mg/m3 were estimated to be 153, 162, and 225 μg vanadium, respectively. There was little difference in the
Relevance of carcinogenic effects / potential:
A clear evidence of carcinogenic activity was observed in the 2-year study in male and female mice. The genetic toxicology studies ( Salmonella typhimurium gene mutations and micronucleated erythrocytes mouse peripheral blood in vivo) showed negative results for mutagenic effects.
Dose descriptor:
LOAEC
Effect level:
1 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: incresaed incidences of alveolar/bronchiolar neoplasms
Remarks on result:
other: Effect type: carcinogenicity (migrated information)

Chamber concentration uniformity was acceptable throughout the 16-day special studies and 2-year studies.

Conclusions:
The survival rate 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. There was clear evidence of carcinogenic activity of vanadium pentoxide in the 2-year study with male and female 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.
Reason / purpose:
reference to other study
Reason / purpose:
reference to other study
Reference
Endpoint:
carcinogenicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well-documented publication
Reason / purpose:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
The National Toxicology Program (NTP) chronic inhalation bioassay of vanadium pentoxide produced ‘‘some’’ and ‘‘equivocal’’ evidence in male and female F344/N rats, respectively (please refer to the study mentioned in the field "Cross-reference to other study" below). No significant pairwise differences or trends with vanadium pentoxide concentration in male or female rat poly-3-adjusted tumor incidence were reported. The ‘‘some’’ and ‘‘equivocal’’ evidence descriptors arose from comparisons of vanadium pentoxide-exposed group incidence rates with NTP-2000- and NIH-07-fed historical control (HC) group incidence ranges. NTP acknowledged that use of data from NIH-07-fed historical control groups could be inappropriate because the vanadium pentoxide study used the NTP-2000 diet, but few studies using this newer diet were available then. The authors of this publication supplemented the early NTP-2000 diet historical control data with data from 25 additional NTP-2000 diet studies conducted subsequent to the vanadium pentoxide bioassay. This widened the historical control tumor incidence ranges.
GLP compliance:
not specified
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Diet: NTP-2000 diet
Route of administration:
inhalation
Vehicle:
not specified
Details on exposure:
no data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
no data
Frequency of treatment:
no data
Post exposure period:
no data
No. of animals per sex per dose:
no data
Control animals:
yes, plain diet
Details on study design:
no data
Positive control:
no data
Observations and examinations performed and frequency:
no data
Sacrifice and pathology:
no data
Other examinations:
The following method was applied:

Incidence rate data for alveolar and bronchiolar (A/B) neoplasms (adenoma, carcinoma, and adenoma or carcinoma) in control group rats fed the NTP-2000 diet were taken from NTP Technical Report 507 (NTP, 2002)* and the NTP historical controls report of May 2011 (NTP, 2011)*. Potential heterogeneity of the two groups of historical control incidence rates was assessed using the Kolmogorov–Smirnov (K–S) 2-sample test (Daniel, 1978; Harter and Owen, 1970)*. This non-parametric statistical test: (1) makes no distributional assumptions, and (2) is responsive to differences in dispersion as well as central tendency. The K–S 2-sample test compares the largest observed absolute difference between the two sample cumulative distribution functions (CDFs) being considered to the test’s critical values that are associated with various discrete and exact p-values.
When significant heterogeneity was shown to be absent, the two groups of historical control incidence rate data were pooled and used to construct new incidence ranges against which the neoplastic findings in the treated groups from the vanadium pentoxide bioassay could be compared. In addition, goodness-of-fit of the binomial distribution to the pooled historical control data was assessed using Tarone’s Z statistic (Tarone, 1979)*. This statistical test also provides a means for assessing the extent to which significant extra-binomial variability may be present in the historical control data. Such overdispersion would dictate the use of a more complicated parametric representation of the sample cumulative distribution function, perhaps as a beta-binomial distribution, which allows explicitly for overdispersion. When appropriate, the fitted binomial distributions were used to estimate the probability of occurrence of tumor incidence rates at least as large as those observed in the concurrent control groups from the vanadium pentoxide bioassay.

*References
- NTP. 2002. NTP Technical Report on the Toxicology and Carcinogenicity Studies of Vanadium Pentoxide (CAS NO. 1314-62-1) in F344/N Rats and B6C3F1 Mice (Inhalation Studies). National Toxicology Program, Research Triangle Park NC. NTP TR 507, NIH Publication No. 03-4441, pp. 352, dated December 2002.
- NTP. 2011. NTP Historical Controls Report – All Routes and Vehicles – F344/N Rats. National Toxicology Program, Research Triangle Park NC. pp. 85, dated May 2011.
- Daniel, W.W., 1978. Applied Nonparametric Statistics. Houghton Mifflin Company, Boston MA, 503 pp.
- Harter, H.L., Owen, D.B., 1970. Selected Tables in Mathematical Statistics - Volume 1. Markham Publishing Company, Chicago IL, 405 pp.
- Tarone, R.E., 1979. Testing the goodness of fit of the binomial distribution. Biometrics 66, 585–590.
Statistics:
Please refer to the field "Other examinations" above
Details on results:
- There is no significant difference in central tendency or dispersion between the two samples of NTP-2000 diet historical control lung tumor incidence data. It is therefore appropriate to combine the two sets of NTP-2000 historical control data.
- Common tumor incidence rate cumulative distribution functions were estimated by pooling the two groups of sample historical control incidence rates.
The largest difference in historical control tumor incidence rate ranges occurred for male rats, where the alveolar/bronchiolar carcinoma incidence rate range tripled, expanding from 0–2% using NTP TR 507 historical control data, to 0–6% using the pooled historical control data. For female rats, both the alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma incidence ranges expanded from 0–6% using the NTP TR 507 historical control data, to 0–8% using the pooled historical control data. The remaining historical control tumor incidence rate ranges were the same for the NTP TR 507 historical control data and the pooled diet historical control data.
- The binomial distribution provided an adequate fit to the pooled historical control data except for female alveolar/bronchiolar adenoma (p ~ 0.01) and possibly for female alveolar/bronchiolar adenoma or carcinoma (p ~ 0.05). For these two endpoints, there is too much dispersion among the sample incidence rates for them to have plausibly arisen by random sampling from a common binomial distribution with a fixed tumor occurrence probability p.
- Given the adequacy of the binomial distribution fits to the male rat historical control data for alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma, it is instructive to use the fitted distribution, i.e., the estimated binomial parameter p, to estimate the probability of obtaining incidence rates, by chance alone, that are at least as large as those in the concurrent male rat control group from the vanadium pentoxide bioassay.
- For male rat alveolar/bronchiolar adenoma, the concurrent control incidence was 4/50, and the estimated probability of an outcome at least this large is 0.0523. For male rat alveolar/bronchiolar adenoma or carcinoma, the concurrent control incidence was also 4/50, and the estimated probability of an outcome at least this large is 0.1141. For comparison, the corresponding nonparametric pooled historical control sample probabilities of these or larger outcomes are 3/34 = 0.0882 and 5/34 = 0.1471, respectively.
- While these probabilities are not sufficiently small to conclude categorically that the concurrent control group of male rats is a statistical ‘‘outlier’’, they are small enough to raise serious concerns that all of the male rats utilized in the vanadium pentoxide bioassay may have had elevated risks of developing alveolar/bronchiolar adenomas even in the absence of vanadium pentoxide exposure. This would readily account for the fact that the unadjusted low concentration male alveolar/bronchiolar adenoma incidence rate (8/49) and the unadjusted low and high concentration male alveolar/bronchiolar adenoma or carcinoma incidence rates (10/49 and 9/50, respectively) exceed the high end of the historical control incidence rate ranges (0–12% and 0–14%, respectively) for these tumors. It would also explain the absence of any statistically significant trend in tumor incidence with increasing vanadium pentoxide exposure. If the entire lot of male rats employed in the vanadium pentoxide bioassay was particularly susceptible to the development of alveolar/bronchiolar adenomas irrespective of vanadium pentoxide exposure, then a valid assessment of the potential carcinogenicity of vanadium pentoxide in male rats could only be achieved by employing pairwise comparisons and concentration-related trend tests that utilize the tumor incidence rates for the concurrent controls.
Conclusions:
The data analysis indicates that there is (1) an absence of any statistically significant differences in tumor incidence between vanadium pentoxide-treated and concurrent control group male and female rats, (2) weakened evidence from comparisons with the widened historical control tumor incidence ranges that result from use of updated historical control data, (3) the likelihood that all of the male rats utilized in the vanadium pentoxide bioassay may have had elevated risks of developing alveolar/bronchiolar adenoma even in the absence of vanadium pentoxide exposure, (4) an extrapolation uncertainty arising from the large discrepancy between the mouse and rat carcinogenic responses to comparable vanadium pentoxide exposures in the NTP bioassay, and (5) an absence of compelling evidence to support a genotoxic mode of action for vanadium pentoxide.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2002

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 50 male and 50 female F344/N rats (approx. 6-7 weeks old) were exposed to V2O5 aerosols at concentrations of 0, 0.5, 1 or 2 mg/m3 by inhalation, 6 hours/d, 5 d/wk, for 104 weeks. The body weight was controlled initially and body weight and clinical finding were recorded every 4 weeks (until week 89) and every 2 weeks from week 92 on. Animals were observed twice daily. Necropsy was performed at study end.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Name of test material (as cited in study report): Vanadium pentaoxide
- Substance type: technical product
- Physical state: solid
- Analytical purity: ca. 99 %
- Impurities (identity and concentrations): XRD analyses of both lots indicated the presence of vanadium pentoxide with no detectable contaminants. No impurities > 1% .
- Lot/batch No.: Lot 1210490 was used in the 16-day and 3-month studies. Lot 1210140 was used in the 16-day special studies and the 2-year studies.
- Stability under test conditions: stability was monitored throughout the study. No degradation of the bulk chemical was detected.
- Other:
Vanadium pentoxide was obtained from Shieldalloy Metallurgical Corporation (Newfield, NJ) in two lots (1210490 and 1210140).
Lot 1210490, was identified as vanadium pentoxide using X-ray diffraction (XRD) analyses and infrared and ultraviolet/visible spectroscopy and by the study laboratory using infrared spectroscopy.
Lot 1210140 was identified using infrared and ultraviolet/visible spectroscopy and by the the study laboratory using XRD analysis.
The purity of lot 1210490 was determined using elemental analyses, weight loss on drying, spark source mass spectrometry, energy-dispersive X-ray (EDX) spectroscopy, and potentiometric titration.
The purity of lot 1210140 was determined using weight loss on drying, potentiometric titration, and ICP/AES.
- No further information on test material was stated.

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: average age: 6 or 7 weeks old on the first day of the study
- Weight at study initiation: average body weight for week 1: 134-136 g (males) and 130-105 g (females)
- Housing: housed individually; stainless steel wire bottom (Hazleton Systems, Inc., Aberdeen, MD); cages and racks were rotated weekly.
- Diet: ad libitum, except during exposure periods; NTP-2000 pelleted diet (Zeigler Brothers, Inc., Gardeners, PA), changed weekly
- Water: ad libitum; tap water (Richland, WA, municipal supply water used) via automatic watering system
- Acclimation period: quarantined for 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 23.9± ca.2°C (75° ± 3° F)
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day

IN-LIFE DATES: From: 1997-01-06 (first exposure) to 1999-01-4/8 (necropsy date)

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: MMAD = 1.0-1.3 µm, GSD = 2.3-2.8
Details on inhalation exposure:
- The generation and delivery system used in the 16-day special studies and the 2-year studies consisted of a linear dust feeder, a particle attrition chamber, and an aerosol distribution system. The linear dust feeder, a slide-bar dust-metering device, was composed of a shuttle bar, body, outlet port, and hopper. As the compressed-air-driven shuttle bar slid back and forth during generation, the metering port aligned with the hopper, which served as a reservoir for the bulk chemical, and was filled with a small amount of vanadium pentoxide powder. As the shuttle bar slid to the dispersing position, the metering port aligned with a compressed-air port in the body and a puff of air from this port dispersed the vanadium pentoxide into the particle attrition chamber. Generator output was regulated by adjusting the cadence of the shuttle bar. The particle attrition chamber used low fluid energy from an air jet tangential to the chamber to deagglomerate the vanadium pentoxide particles. After deagglomeration, the particles were swept into a classification zone where smaller particles exited to the distribution line; larger particles were thrown to the perimeter of the classifier by centrifugal force and were reentrained into the impacting air jet, and the process was repeated until the particles were sufficiently deagglomerated. The aerosol passed through the distribution lines to the exposure chambers. A pneumatic pump designed by the study laboratory was located at each chamber inlet and drew aerosol from the distribution line into the chamber inlet, where it was diluted with conditioned air to the appropriate concentration. Flow through the distribution line was controlled by Air-Vac pumps (Air-Vac Engineering, Milford, CT), and pressure was monitored by photohelic differential pressure gauges (Dwyer Instruments, Inc., Michigan City, IN).
- The stainless steel chambers (Lab Products, Inc., Harford Systems Division, Aberdeen, MD), were designed so that uniform aerosol concentrations could be maintained throughout the chambers when catch pans were in place. The total active mixing volume of each chamber was 1.7 m³.

CHAMBER ATMOSPHERE CHARACTERIZATION
- The particle size distribution in each chamber was determined prior to the start of all studies, during the first week of the 16-day and 3-month studies, during the first 2 weeks of the 2-year studies, and monthly during the 3-month and 2-year studies.
- For the 16-day special studies and the 2-year studies, a Mercer-style seven-stage impactor was used. The stages (glass coverslips lightly sprayed with silicon) were analyzed by ICP/AES, and the relative mass collected on each stage was analyzed by probit analysis.

OTHER
- The uniformity of aerosol concentration in the inhalation exposure chambers without animals was evaluated before each of the studies began; concentration uniformity with animals present in the chambers was also measured. During the 16-day and 3-month studies, minor excursions in chamber uniformity values were observed in one or more exposure chambers, but these excursions had no impact on the studies. Chamber concentration uniformity was acceptable throughout the 16-day special studies and 2-year studies.
- The stability of vanadium pentoxide in the exposure system was tested with XRD analysis. XRD analyses indicated no detectable build-up of degradation products at a detection limit of approximately 1%.



Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- During all studies, chamber aerosol concentrations were monitored with real-time aerosol monitors (RAMs) that used a pulsed-light-emitting diode in combination with a silicon detector to sense light scattered over a forward angular range of 45° to 95° by particles traversing the sensing volume. The instruments respond to particles 0.1 to 20 μm in diameter.
- For the 16-day special studies and the 2-year studies, the sampling system consisted of a valve that multiplexed each RAM to two or three exposure chambers and to a HEPA filter and/or the control chamber or room; selection of sampling streams and data acquisition from each RAM was remotely controlled by a computer. Equations for calibration curves were stored in the computers and were used to convert the measured voltages to exposure concentrations.
- Each RAM was calibrated daily during the 16-day and 3-month studies by correlating the measured voltage with vanadium pentoxide concentrations determined by gravimetric analysis of glass fiber filters and one to two times per week during the 2-year studies by ICP/AES or ICP/mass spectrometry analysis of Pallflex® TX40H120WW glass fiber filters.
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
6 hours per day, 5 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
0.5 mg/m³ air (nominal)
Dose / conc.:
1 mg/m³ air (nominal)
Dose / conc.:
2 mg/m³ air (nominal)
No. of animals per sex per dose:
core study: groups of 50 male and 50 female rats
tissue burden analyisis: 40 female rats per exposed group; separate control groups of 15 female rats were used as chamber controls
Control animals:
yes
Details on study design:
- Dose selection rationale: based on the incidences and severities of respiratory lesions and increased lung weights in male and female rats, concentrations of 4 mg/m3 or greater were considered to be too high for use in a 2-year study. The exposure concentrations selected for the 2-year inhalation study in rats were 0.5, 1, and 2 mg/m3.
- Rationale for animal assignment (if not random): randomly into groups of approximately equal initial mean body weights.
Positive control:
not stated

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- The health of the animals was monitored during the studies according to the protocols of the NTP Sentinel Animal Program

DETAILED CLINICAL OBSERVATIONS: Yes
- Clinical findings were recorded every 4 weeks from week 5 through 89 and every 2 weeks from week 92 until the end of the studies

BODY WEIGHT: Yes
- Time schedule for examinations: body weights were recorded on day 1 and every 4 weeks from week 5 through 89 and every 2 weeks from week 92 until the end of the studies

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

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 data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER:
- Tissue Burden Studies: groups of five female rats were evaluated on days 1, 5, 12, 26, 54, 173, 360, and 542;
- Total lung weight, right lung burden, and left lung histopathology were measured in exposed animals at all time points.
- Blood vanadium concentration was measured in all animals at all time points after day 12. Groups of five chamber control animals were bled at each of these time points and returned to their chambers and used for subsequent bleedings. Blood was obtained by cardiac puncture from exposed animals or from the retroorbital sinus of chamber control animals.
Sacrifice and pathology:
Method of sacrifice: CO2 asphyxiation.

PATHOLOGY: Yes
- Necropsy was performed at study end on all core study animals.
- All organs and tissues were examined for grossly visible lesions, and all major tissues were prepared for microscopic examination.

HISTOPATHOLOGY:
- Complete histopathology was performed on all core study animals. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gallbladder (mice only), heart and aorta, large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum, and ileum), kidney, larynx, liver, lung and mainstem bronchi, lymph nodes (mandibular, mediastinal, mesenteric, and bronchial), mammary gland (except male mice), nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea,
urinary bladder and uterus.
Other examinations:
Five male and five female rats were randomly selected for parasite evaluation and gross observation of disease.
Statistics:
Survival Analyses:
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. Animals found dead of other than natural causes were censored from the survival analyses; animals dying from natural causes were not censored. Statistical analyses for possible doserelated effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.

Analysis of Continuous Variables:
Two approaches were employed to assess the significance of pairwise comparisons between exposed and control groups in the analysis of continuous variables. Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Hematology, clinical chemistry, urinalysis, urine concentrating ability, cardiopulmonary, immunotoxicologic, cell proliferation, tissue concentrations, spermatid, and epididymal spermatozoal data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test). Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973). Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across exposure concentrations. (for more information see publication)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
only marginally less for the 2 mg/m3–exposed females
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Based on the analysis of Starr et al. (2012), effects observed in the male rat are not significant.
Other effects:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
- Survival of the animals were similar to the controls

BODY WEIGHT AND WEIGHT GAIN
- Body weights of the animals were similar to the controls except body weight of the 2 mg/m3–exposed females which was less

HISTOPATHOLOGY: NON-NEOPLASTIC
- Non-neoplastic lesions occurred in respiratory system of males and females (lung, larynx, and nose) , and the severities of these lesions generally increased with increasing exposure concentration.

LUNGS:
- Effects in males: alveolar epithelium, hyperplasia (7/50, 24/49, 34/48, 49/50); bronchiole, epithelium hyperplasia (3/50, 17/49, 31/48, 49/50); alveolar epithelium, metaplasia, squamous (1/50, 0/49, 0/48, 21/50); bronchiole, metaplasia, squamous (0/50, 0/49, 0/48, 7/50); inflammation, chronic active (5/50, 8/49, 24/48, 42/50); interstitial, fibrosis (7/50, 7/49, 16/48, 38/50); alveolus, infiltration cellular, histiocyte (22/50, 40/49,45/48, 50/50).
- Effects in females: alveolar epithelium, hyperplasia (4/49, 8/49, 21/50, 50/50); bronchiole, epithelium hyperplasia (6/49, 5/49, 14/50, 48/50); alveolar epithelium, metaplasia, squamous (0/49, 0/49, 0/50, 6/50); inflammation, chronic active (10/49, 10/49, 14/50, 40/50); interstitial, fibrosis (19/49, 7/49, 12/50, 32/50); alveolus, infiltration cellular, histiocyte (26/49, 35/49, 44/50, 50/50).
- Incidences of minimal to mild chronic active inflammation and interstitial fibrosis in the lungs were significantly increased in males exposed to 1 or 2 mg/m3 and females exposed to 2 mg/m3, and the incidences of histiocytic cellular infiltrate of the alveolus were increased in all exposed groups of males and females. The inflammatory lesions were primarily minimal to mild and consisted of interstitial and perivascular infiltrates of mostly mononuclear inflammatory cells that were occasionally within alveoli. Alveolar septa were occasionally thickened by thin strands of eosinophilic fibrillar material (fibrosis). The histiocytic infiltrate was also minimal to mild, consisting of scattered intraalveolar macrophages that contained large amounts of foamy intracytoplasmic material, interpreted as pulmonary surfactant. Additionally, scant amounts of eosinophilic material (surfactant) similar to that observed within alveolar macrophages was also free within alveoli; however, a separate diagnosis was not made. A brownish pigment (pigmentation) was visible in alveolar macrophages in some males and females exposed to 2 mg/m3 and in females exposed to 1 mg/m3; it was a mild change considered of little biological significance and was not further characterized.

LARYNX:
- Effects in males: inflammation, chronic (3/49, 20/50, 17/50, 28/49); respiratory epithelium, epiglottis degeneration (0/49, 22/50, 23/50, 33/49); respiratory epithelium, epiglottis, hyperplasia (0/49, 18/50, 34/50, 32/49); respiratory epithelium, epiglottis, metaplasia, squamous (0/49, 9/50, 16/50, 19/49).
- Effects in females: inflammation, chronic (8/50, 26/49, 27/49, 37/50); respiratory epithelium, epiglottis degeneration (2/50, 33/49, 26/49, 40/50);
respiratory epithelium, epiglottis, hyperplasia (0/50, 25/49, 26/49, 33/50); respiratory epithelium, epiglottis, metaplasia, squamous (2/50, 7/49, 7/49, 16/50).
- There were increased incidences of minimal to mild lesions of the larynx in exposed males and females. The incidences generally increased with increasing exposure concentration and included chronic inflammation of the larynx and degeneration, hyperplasia, and squamous metaplasia of the respiratory epithelium of the epiglottis. The inflammation consisted of a mixture of mononuclear and granulocytic inflammatory cells in the submucosa beneath the epithelium lining the base of the epiglottis, ventral pouch, and caudal larynx. The degeneration of the respiratory epithelium was characterized by a loss or decrease in the height of cilia and shortening of the normally columnar to cuboidal surface epithelial cells lining the laryngeal surface of the base of the epiglottis. Squamous metaplasia was diagnosed when the ciliated cells were replaced by one or more layers of flattened squamous epithelium. In the same area, the respiratory epithelium was mildly thickened in many animals; this change was diagnosed as hyperplasia. These changes are relatively minimal, commonly occur in rats in NTP inhalation studies, and represent a common response to laryngeal injury.

NOSE:
- Effects in males: goblet cell, respiratory epithelium, hyperplasia (4/49, 15/50, 12/49, 17/48)
- Effects in females: goblet cell, respiratory epithelium, hyperplasia (13/50, 18/50, 16/50, 30/50)
- There were increased incidences of mild goblet cell hyperplasia of the nasal respiratory epithelium in all groups of exposed male rats and in females exposed to 2 mg/m3. Increased numbers of goblet cells were most notable in the respiratory epithelium lining the median septum adjacent to the area of the vomeronasal organ.

KIDNEY:
- The incidences of nephropathy were significantly increased in male rats exposed to 1 or 2 mg/m3. Nephropathy is a common lesion in aged rats, particularly males, and has been diagnosed in virtually all males in NTP 2-year studies that used the NIH-07 diet. In those studies, chemical exacerbation of nephropathy was identified by increased severity. With the NTP-2000 diet, the severity of spontaneous nephropathy has been reduced. In this study, the severity of nephropathy was not increased in exposed groups of males. Also, exposed females were not affected. Although the NTP doesn’t have a formal historical control database for nonneoplastic lesions, a review of recent studies indicates that the incidence in the male chamber control group in the current study is low. It is not clear if the increased incidences in this study were related to exposure to vanadium or were a reflection of the low incidence in the control group. Regardless, nephropathy was a relatively weak response and was likely of marginal biological significance.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)

Please note that the following carcinogenic effects as reported in the original study are not statistically significant according to Starr et al. (2012).

LUNG:
- Effects in males: alveolar/ bronchiolar adenoma (4/50, 8/49, 5/48, 6/50); alveolar/bronchiolar carcinoma (0/50, 3/49, 1/48, 3/50); alveolar/ bronchiolar adenoma or carcinoma (4/50, 10/49, 6/48, 9/50)
- Effects in females: none (equivocal findings: alveolar/bronchiolar adenoma (0/49, 3/49, 1/50, 0/50); alveolar/bronchiolar adenoma or carcinoma (0/49, 3/49, 1/50, 1/50)
- Alveolar/bronchiolar neoplasms were present in exposed groups of males and one 2 mg/m3 female. Alveolar/bronchiolar adenomas were present in 0.5 and 1 mg/m3 females. Additionally, one female exposed to 2 mg/m3 had an alveolar/bronchiolar carcinoma. There were no statistically significant increases in the incidences of lung neoplasms in rats.
- There were increased incidences of alveolar epithelial hyperplasia and bronchiole hyperplasia in the lungs of males exposed to 0.5 mg/m3 or greater and females exposed to 1 or 2 mg/m3. The severities of these lesions were increased in 2 mg/m3 males and females. In affected animals, this was essentially a diffuse change with proliferation of epithelium in the distal terminal bronchioles and immediately associated alveolar ducts and alveoli. Normally flattened epithelium was replaced with cuboidal epithelium.
- Increased incidences of squamous metaplasia of the alveoli occurred in male and, to a lesser extent, in female rats exposed to 2 mg/m3. There were a spectrum of changes ranging from minimal to severe. Minimal lesions were characterized by a single alveolus with the thin type I cells which normally line alveoli replaced by one to several layers of squamous epithelium. Severe lesions were much larger, often involving an area approximately 1 cm in diameter. Many alveoli were involved and there was apparent coalescence of the metaplasia. There were also lesions of intermediate severity. Keratin production was a prominent feature of the squamous metaplasia observed in this study. Keratin often filled the affected alveoli, and in some of the lesions, cyst-like structures filled with keratinous material were formed. In a few animals (predominantly males), the squamous metaplasia extended into the distal airways and was diagnosed as bronchiole squamous metaplasia. Commonly dispersed within the squamous lesions were areas of respiratory epithelial metaplasia in which the alveolar epithelium was replaced by tall cuboidal to columnar epithelium with cilia often present and with mucous material filling the alveolar lumen.

UTERUS:
- The incidences of stromal polyp occurred with a positive trend in female rats (chamber control, 6/50; 0.5 mg/m3, 3/50; 1 mg/m3, 7/50; 2 mg/m3, 13/50). However, the incidence in the 2 mg/m3 group was within the historical range in controls. Endometrial stromal polyps are common neoplasms in the F344/N rat in NTP studies. They are benign neoplasms and generally do not progress to malignancy; however, they occasionally do progress to stromal sarcoma. In this study, when the incidences of stromal polyp were combined with the single incidence of stromal sarcoma, the combined incidence in 2 mg/m3 females was significantly increased. The marginal increase in the incidence of stromal polyp and stromal sarcoma (combined) in females exposed to 2 mg/m3 was not considered related to exposure to vanadium pentoxide.

OTHER FINDINGS
LUNG BURDEN STUDIES:
- Histopathology: the left lung lobe from each animal was infused with 10% neutral buffered formalin, and sections were examined microscopically. The purpose was to follow progression of the lung lesions. Following day 1 of exposure, there was an infiltrate of alveolar macrophages in the lungs. With continued exposure, increased numbers of alveolar macrophages, interstitial mononuclear inflammatory cell infiltrates, and hyperplasia of alveolar and bronchiolar epithelium were observed. In rats exposed to 2 mg/m3, there was an increase in severity of the hyperplasia between days 54 and 173. An increase in severity was not obvious between days 173 and 360, but hyperplasia appeared more severe on day 542. Hyperplasia was observed in only a few animals exposed to 1 mg/m3 and only on day 542. The minimal fibrosis observed in the 2-year study was not readily apparent on day 542 or earlier.
- Lung weights from exposed female rats increased throughout the study. Although there appeared to be an exposure concentration-related increase in lung weights after day 26 of the study, it was primarily due to increases in lung weights of female rats exposed to 2 mg/m3. In general, lung weights of 0.5 or 1 mg/m3 females were similar.
- Lung burden data appeared proportional to exposure concentration in rats.
- Though deposition patterns were similar between rats and mice, the maximum lung burdens occurred at day 173 in rats. The lung burdens appeared to reach steady state at the lowest exposure concentrations (0.5 mg/m3). A decline in lung burdens was observed. The retention of vanadium in the lungs at 18 months was ca. 13% to 15% in rats. The total lung doses for rats exposed to 0.5, 1, or 2 mg/m3 were estimated to be 130, 175, and 308 μg vanadium, respectively.
- Lung clearance half-times were considerably longer than those observed in the 16-day special studies.

BLOOD:
- Vanadium was detected in the blood at concentrations several orders of magnitude lower than those measured in the lungs of exposed rats, and blood vanadium concentrations in exposed groups were only marginally increased over that of the chamber control group. Overall, blood vanadium concentrations appeared to increase with increasing exposure concentration; however, this proportionality was less clear when the 0.5 and 1 mg/m3 groups were compared.
- Blood vanadium concentrations in all exposed groups appeared to peak on days 26 or 54 after which there was a decline throughout the rest of the study. This response was similar to that seen in lung burdens. However, these changes in concentrations were small, making it difficult to determine if there was an increase in elimination of vanadium from the blood or a decreased absorption from the lung due to reduced deposition, especially at the higher exposure concentrations.

Effect levels

Dose descriptor:
LOAEC
Remarks:
local
Effect level:
0.5 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Chamber concentration uniformity was acceptable throughout the 16-day special studies and 2-year studies.

Applicant's summary and conclusion

Conclusions:
Survival rates and body weights were not affected in rats exposed to vanadium pentoxide for 2 years. As in the 3-month studies, the respiratory tract was the primary site of toxicity in rats. Under the conditions of this 2-year inhalation study, some evidence of carcinogenic activity of vanadium pentoxide in male F344/N rats and equivocal evidence of carcinogenic activity of vanadium pentoxide in female F344/N rats was reported. Based on the analysis of Starr et al. (2012), the observed carconogenic effects are statistically not significant as follows:
(1) there are not any statistically significant differences in tumor incidence between vanadium pentoxide-treated and concurrent control group male and female rats,
(2) there is weakened evidence from comparisons with the widened historical control tumor incidence ranges that result from use of updated historical control data, and
(3) there is a likelihood that all of the male rats utilized in the vanadium pentoxide bioassay may have had elevated risks of developing alveolar/bronchiolar adenoma even in the absence of vanadium pentoxide exposure.
The genetic toxicology studies ( Salmonella typhimurium gene mutations and Micronucleated erythrocytes Mouse peripheral blood in vivo) show negative results for mutagenic effects.
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 rats and an unusual squamous metaplasia of the lung in male and female rats. The lowest concentration tested (0.5 mg/m3) represents a LOAEC for local effects in the respiratory tract.