Vanadium

Specific investigations: other studies

Administrative data

Endpoint:

mechanistic studies

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

2009-05-07 to 2010-04-30

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study reliable without restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Divanadium pentaoxide was administered by nose-only inhalation to female mice for a period of 16 days (6 h/day) at target concentrations of 0.25, 1.0 and 4.0 mg/m3 test item in air. Female B6C3F1/Hsd mice were allocated to 4 groups of 48 mice each. The mice of group 1 served as air controls. The mice of each group were further allocated in seven subgroups to evaluate a range of specific toxicological end-points in the lungs and to obtain data on the concentration of vanadium in blood and lungs. One additional group of six mice (group 5) was used as positive controls for the comet assay subgroup.

Throughout the treatment period all mice were observed daily for viability and clinical signs. In addition, the body weight of each mouse was recorded weekly during the acclimatization and treatment period. After completion of treatment, animals of each subgroup were subjected to determination of vanadium in blood and lungs, to necropsy, to weighing of the lungs, to lung preparation for cell proliferation analysis after 7 and 16 days of treatment, respectively, to biomarker analysis in lung tissues, to investigations on DNA lesions in the lungs and/or to a comet assay with BAL and pulmonary cells.

GLP compliance:

yes

Type of method:

in vivo

Endpoint addressed:

repeated dose toxicity: inhalation

Test material

Test material information

Constituent 1

Reference substance name:

Divanadium pentaoxide

EC Number:

215-239-8

EC Name:

Divanadium pentaoxide

Cas Number:

1314-62-1

Molecular formula:

V2O5

IUPAC Name:

divanadium pentaoxide

Details on test material:

- Name of test material (as cited in study report): Vanadium pentoxide
- Molecular weight: 182 g/mol
- Physical state: solid, yellow-orange
- Storage condition of test material: at room temperature (range of 20 +/- 5 °C) under dry conditions.
No further information on the test material was stated.

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories Ltd., 5190 Dominion Drive, Dublin, VA24084, U.S.A.
- Age at study initiation (Treatmetn Day 1): group 1 to 4: 9 to 10 weeks; group 5: 11 to 12 weeks .
- Weight at study initiation (Treatment Day 1): group 1: 21.8 +/- 1.1 g; group 2: 21.5 +/- 1.3 g; group 3: 21.8 +/- 1.1 g; group 4: 21.4 +/- 1.3; group 5: 21.1 +/- 0.2 g.
- Housing: individually in Makrolon® type-2 cages with wire mesh tops and standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & Co, KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland).
- Diet (ad libitum): pelleted standard Kliba Nafag 3433 (batch no. 76/08) mouse maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland)
- Water (ad libitum): community tap-water from Itingen.
- Acclimation period: at least 7 days under laboratory conditions, after clinical health examination. Only mice without any visible signs of illness were used for the study. The mice of groups 1 to 4 were accustomed to the restraint tubes and the exposure conditions for 3 daily periods of approximately 1, 3 and 5 hours, respectively.

ENVIRONMENTAL CONDITIONS
Optimal Hygienic Conditions behind a barriersystem in an air-conditioned facility.
- Temperature: 22 ± 3 °C
- Relative humidity: 30 - 70%
- Air changes: approximately 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12 (Music during the light cycle.)
No further information on the test animals was stated.

Administration / exposure

Route of administration:

inhalation: dust

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus/Method of holding animals in test chamber: inhalation exposure was performed using a system similar to that originally described by Sachsse et al. ( 1976). The mice are confined separately in restraint tubes which are positioned radially around the flow-past, nose-only exposure chamber described by Cannon et al. (1983). The design of this chamber is based upon the fluid dynamic modeling of the test aerosol flow.
The exposure system ensured a uniform distribution and provided a constant flow of test material to each exposure tube. The flow of air at each tube was 0.5 L/min, which was sufficient to minimize re-breathing of the test aerosol as it was more than twice the respiratory minute volume of a mouse.
Before commencement of the exposure of the group(s), technical trials (procedures based on GLP) were conducted (without mice) using the inhalation system foreseen for the study.

- System of generating particulates/aerosols: a dust aerosol was generated from the test item using a rotating brush aerosol generator connected to a micronizing jet mill. The aerosol generated was then discharged into the exposure chamber through a 63Ni charge neutraliser. An airvacuum dilution system was used to achieve the target aerosol concentration for groups 2 and 3. Air control mice (group 1) were exposed to air under the same conditions as mice treated with the test item.
The air for aerosol generation was delivered by a model 'Atlas Copco ZT30-8' oil-free, rotating gear-wheel compressor system'. Additionally, there was an air-cleaning filter system mounted at the entry in the exposure room.

- Temperature, humidity, pressure in air chamber: oxygen concentrations in the chamber were measured continuously during each exposure using a calibrated device. The oxygen concentration was maintained above 19% during the exposure period. The results were reported three times during each daily exposure.
The relative humidity and temperature in the chamber were measured continuously during each exposure using a calibrated device. The results were reported three times during each daily exposure.

Results on temperature/humidity/oxygen concentration:
Group 1: 22.7 ± 0.3 °C; 1.0 ± 0.3 %; 20.7 ± 0.0%
Group 2: 22.1 ± 0.1 °C; 3.8 ± 0.1 %; 20.6 ± 0.0 %
Group 3: 21.8 ± 0.1 °C; 1.9 ± 0.8 %; 20.6 ± 0.0 %
Group 4: 21.2 ± 0.1 °C; 0.7 ± 0.6 %; 20.4 ± 0.0 %

- Air flow rate: measured for the collection of samples for the determination of test item concentration and particle size using a dry-test meter (‘Schlumberger Industries SA’, City of Geneva) and/or a pressure gauge (Timeus & Co., Zürich), calibrated with a reference dry-test meter.
The exposure air flow rate was adjusted as appropriate before the start of the exposure using calibrated flow-meters and / or pressure gauges. The actual airflow rates were recorded three times during each daily exposure.

The aerosol concentrations of the test item determined gravimetrically and chemically, particle size distribution determined gravimetrically and chemically, relative humidity, temperature and oxygen concentration, were measured on test aerosol samples collected directly from the delivery tube in the breathing zone of the mice, at an empty port of the exposure chamber.

- Method of particle size determination: distribution of particle size in the generated aerosol was measured by gravimetry twice during the 16-day treatment period in each of groups 3 and 4 using a cascade impactor. In group 3, sampling was performed over a period of 4 consecutive days. Following gravimetric determination of the particulate material on each impactor screen, the impactor samples were analyzed chemically. The samples were analyzed for vanadium using an AAS method and reported as vanadium pentoxide after recalculation.

Results of gravimetric determination of MMAD (GSD):
Group 3: 1.22 - 1.26 µm (1.89 - 1.92)
Group 4: 1.24 - 1.43 µm (1.89 - 1.92)

Determination of particle size distribution in group 2 was not feasible due to the low aerosol concentration. However, the MMAD for group 2 was considered to be similar to the other groups because one single aerosol generation system was used for all three treatment groups. The MMADs were at the lower limit of the target range of 1 to 3 μm, therefore deposition of the particles can be assumed to have occurred mainly in the lower but also in the upper respiratory tract.

Chemical determination of MMAD (GSD):
Group 3: 1.27 - 1.34 µm (1.77 - 1.86)
Group 4: 1.33 - 1.41 µm (1.75 - 1.82)

TEST ATMOSPHERE
- Brief description of analytical method used:
1.) Gravimetrical determination: once daily for groups 2 to 4 using Millipore® durapore filters, Type HVLP, (Polyvinylidenedifluoride membrane, pore size 0.45 μm) loaded in a 47 mm in-line stainless steel filter sampling device (Gelman Science Inc., Ann Arbor, Michigan / U.S.A.).
2.) Chemical determination: vanadium pentoxide concentrations in the generated aerosol were performed once daily for groups 2 to 4 using the filters collected for gravimetric determinations. After weighing, filter samples and a blank filter were transferred to appropriately labeled vials. The samples were analyzed for vanadium using an atomic absorption spectroscopy (AAS) method and reported as vanadium Pentoxide after recalculation.

NOMINAL DETERMINATION OF AEROSOL CONCENTRATION
The test item usage was measured by weighing the generator cylinder containing the test item before and after exposure to determine the quantity of test item used. The weight used was then divided by the total air-flow volume to give the nominal concentration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Please refer to "Details on exposure" above

Duration of treatment / exposure:

6 hours daily 16 consecutive days (except subgroup B: 7 consecutive days)

Frequency of treatment:

daily

Post exposure period:

no

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.246 ± 0.026, 0.993 ± 0.122, 4.01 ± 0.55 mg/m^3
Basis:
other: chemical aerosol concentration

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 0.25, 1.0 and 4.0 mg/m^3 air
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0.231 ± 0.070, 1.03 ± 0.12, and 4.02 ± 0.45 mg/m^3
Basis:
analytical conc.

No. of animals per sex per dose:

Group 1 (air control): 48 females
Group 2 (0.25 mg/m^3 air): 48 females
Group 3 (1.0 mg/m^3 air): 48 females
Group 4 (4.0 mg/m^3 air): 48 females
Group 5 (positive control for comet assay treated with methylmethansulfonate ): 6 females

Control animals:

yes, concurrent vehicle

Details on study design:

Groups 1 to 4 were further divided into 7 subgroups (Group 5 was only a positive control for the Comet assay):
Subgroup A: vanadium pentoxide
Subgroup B: cell proliferation (day 7)
Subgroup C: cell proliferation (day 16), histopathology
Subgroup D: biomarkers in lung tissue (Glutathione, α-tocopherol)
Subgroup E: biomarkers in lung tissue (F2-isoprostanes)
Subgroup F: DNA lesions
Subgroup G: Comet assay
No further details on study design was stated.

Examinations

Examinations:

VIABILITY/MORTALITY
- Twice daily, prior to and following exposure and at least once daily during the quarantine and the acclimatization periods.

CLINICAL SIGNS
- Recorded once weekly during the acclimatization period.
- During 16-day treatment period, at least once daily before exposure. On day 8 of exposure, only the clinical signs of subgroup B were recorded.

BODY WEIGHTS
- Each mouse was weighed approximately weekly during the acclimatization and treatment periods (generally before exposure/dosing, with the exception of subgroups D and E of groups 3 and 4 where the body weights were recorded after exposure on day 9 of exposure).

VANADIUM IN WHOLE BLOOD AND LUNGS (examination performed in subgroup A mice):
- Whole blood: within 5 minutes after the end of the last exposure, blood samples were collected from the vena cava of mice.
- Lungs: removed and weighed.
- All samples were analyzed for vanadium using an atomic absorption spectroscopy (AAS) method validated under Harlan Laboratories Study B34154 and reported as Vanadium.

PATHOLOGY:
- Mice were anesthetized by intraperitoneal injection of pentobarbitone killed by exsanguination and examined for macroscopical findings.
- All surviving mice of allocation B and C were weighed and necropsied after 7 days and 16 days of exposure, respectively.
- All mice surviving to the end of the observation period were and killed.
- Lung weight was recorded at the scheduled dates of necropsy.
- Samples of the lungs were collected and fixed in neutral phosphate buffered 4% formaldehyde solution. The lungs were instilled with the fixative.
- Lungs from all allocation B and C mice were processed, embedded in paraffin wax, cut at a nominal thickness of 4 micrometers. The lung slides were stained for the two endogenous proliferation markers PCNA and Ki67.
- Lung slides of allocation C mice were stained with hematoxylin and eosin.

INVESTIGATION OF CELL PROLIFERATION
-Slides stained for two endogenous proliferation markers (PCNA and Ki67) of all lungs from allocation B (7 days of exposure) and allocation C (16 days of exposure) mice were examined.

HISTOPATHOLOGY
- Slides stained with hematoxylin and eosin of all lungs of allocation C mice (16 days of exposure) were examined. Attempts were made to correlate gross observations with microscopic findings.

INVESATIGATION ON BIOMARKERS IN LUNG TISSUES
One day after the last exposure, the lungs were removed from subgroups D and E mice whilst they were maintained under sodium pentobarbitone anaesthesia AND allocated to the following analyses:
- Subgroup D mice: right lung lobes analyzed for glutathione; left lung lobes analyzed for α-tocopherol.
- Subgroup E mice: whole lung analyzed for F2-Isoprostanes.
- Following sampling, the weight of the lung lobes (subgroup D) or of the whole lung (subgroup E) was recorded.
- These animals were also examined for macroscopical findings.

INVESTIGATION ON DNA LESIONS
As soon as feasible after the last exposure of groups 1, 2, 3 and 4, the following procedures were performed on all mice of subgroup F:
- Lung were perfused through the right ventricle with saline. Afterwards the lungs were intubated via the trachea and lavaged with approx. 20 mL saline. The lungs were then further perfused under resuscitation using saline.
- Lung lobes were excised and shock frozen in liquid nitrogen.
- After completion of the sampling, lung samples were shipped on dry ice for the determination of DNA lesions.

COMEL ASSAY
As soon as feasible after the last exposure of groups 1, 2, 3, and 4, and 4 hours after the treatment of the positive controls (group 5), the following procedures were performed on all mice of subgroup G:
- Lungs were perfused and excised lung lobes were then minced in 1 ml ice-cold mincing buffer using fine scissors. The resulting cell suspension (minced lung tissue cells) was filtered through a 40 μm cell strainer. The cell suspension was then centrifuged at about 5000 g for 1 min and the resulting pellet resuspended in 0.7% agarose. The cells isolated from lung lavage (BronchioAlveolar Lavage (BAL) cells) were centrifuged at 300 xg for 10 min and resuspended in 0.7% agarose.
- Lung samples were analysed for DNA strand breaks using the comet assay.
- Positive control item (Comet assay): Methylmethansulfonate (MMS); purity: 99 %; dissolved in: 0.9 % NaCl solution

Positive control:

Please refer to "Examinations" above.

Results and discussion

Details on results:

VIABILITY AND MORTALITY
- One mouse of group 3 (subgroup C) was found dead shortly after the end of exposure during the second week of treatment.
- This death was considered as incidental and not related to treatment.
- There were no further unscheduled deaths.

CLINICAL SIGNS
- No clinical signs were noted during the course of this study.

BODY WEIGHTS
- Marginal body weight loss was noted in groups 3 and 4 in week 1 (statistically significance in group 4).
- At the end of the treatment period these animals recovered and body weights were similar across all groups.
- There were no effects on the body weight development in group 2.

VANADIUM IN WHOLE BLOOD AND LUNGS
- The analyses were for the concentrations of vanadium in blood and lung tissue and are reported as such, rather than being converted to vanadium pentoxide. Although most of the vanadium in lung will probably have remained as vanadium pentoxide, there are likely changes in oxidation state within this tissue during absorption that were not identified. It is unlikely that any of the vanadium in blood will be present as the pentoxide.
- The concentration of vanadium in the lungs increased dose-proportionally from group 2 to group 3.
- Vanadium values for blood and lung samples in group 4 were higher when compared with group 3, but less than dose proportionally; please refer for results to table 1. in "Any other information on results incl. tables" below.

ORGAN WEIGHTS
- After 16 days of vanadium pentoxide exposure, lung weights and lung weight/body weight ratios showed dose-related increases in groups 3 and 4 (subgroups A, C, D, E); differences to controls were statistically significant.
- At the interim kill after 7 days (subgroup B), there were no effects on the lung weights in group 3 and the statistically significant increased lung weight in group 4 was less pronounced.
- There were no effects on the lung weight in group 2 that were considered to be related to treatment.
- The statistically significant increases in the weight of the right lung lobe, but not the left lung lobe, and in the lung weight/body weight ratios of subgroup D animals were considered to be incidental in the absence of corresponding histopathological findings in this group, and the absence of significant weight changes in the other subgroups that had been simultaneously exposed.

PATHOLOGY
- At necropsy of subgroups A, B, C, D and E animals, no gross findings of the lung were recorded that distinguished test item exposed mice from controls.
- A reddish discolored lung was noted in the animal of group C which prematurely died. This kind of lesion is a common alteration in decedents and, therefore, was considered to be related to a certain degree of tissue autolysis due to delayed necropsy of this animal and not to represent of a primary treatment-related effect.

HISTOPATHOLOGY
- Multifocal/diffuse alveolar histiocytosis and multifocal subacute alveolitis was noted with a dose-related increase in severity in almost all mice of subgroup C in groups 3 and 4.
- In addition, multifocal granulocytic infiltration was noted in 4 out of 6 mice of group 3 and 5 out of 6 mice of group 4.
- Focal alveolar histiocytosis of minimal degree which was noted in 1 mouse of group 2. This finding was considered to be incidental.

CELL PROLIFERATION
Immunohistochemistry (based on the mean numbers of marker-positive cells in animals of subgroup B and C):
- Ki67: mean grades of proliferation rate after 7 or 16 days were time-dependent and dose-related increased in groups 3 and 4. The grades in group 2 were similar to controls.
- PCNA: mean grades of proliferation rate were dose-related increased in groups 3 and 4 after 7 days and time-dependently increased in group 4 after 16 days. The grades in group 2 as well as after 16 days in group 3 were similar to controls.

BIOMARKER DETERMINATION
- Slightly, but statistically significantly reduced mean α-tocopherol concentrations were recorded in the lungs of group 2 animals compared with controls
- Slightly, but statistically significantly increased values were noted for group 4 (subgroup D). No effect was noted in group 3.
- Slightly, but statistically significantly decreased mean concentrations of reduced glutathione (GSH) were observed in the lungs of group 2 compared with controls and no effects were noted in groups 3 and 4 (subgroup D).
-Mean concentrations of oxidised glutathione (GSSG) were statistically significantly increased in groups 2 to 4 (dose-related for groups 2 and 3, and group 4 levels were higher than in group 2).
- Accordingly, the GSH/GSSG ratios were statistically significantly decreased.
- No effects of treatment with the test item on F2-Isoprostane concentrations in the lungs were noted in groups 2 to 4 (subgroup E).

DNA LESIONS
- Quantification of DNA lesions revealed a statistically significant and dose-related increase of 8-oxodGuo in groups 3 and 4.
- Cytosine adduct dCyd341 levels were not altered compared to control group 1.
- Other lesions looked for were below the detection limits of the methods used.

COMET ASSAY
- Analysis of cells obtained from bronchioalveolar lavage or from minced lung tissue of animals of sungroup G showed that treatment of mice with vanadium pentoxide did not induce any DNA damage in the in vivo Comet assay:

Any other information on results incl. tables

Table 1.

Group	Vanadium in whole blood [μg/L]	Vanadium in lung samples [μg/g]
2	- *	8.02 ± 0.84
3	51.15 ± 6.02	31.01 ± 2.22
4	160.13 ± 16.48	64.35 ± 7.45

* below lowest calibration point (13 μg/L)

Applicant's summary and conclusion

Conclusions:

After exposure of groups of female mice by nose-only inhalation to divandium pentaoxide concentrations of 0, 0.25, 1.0 and 4 mg/m3, the levels of biomarkers in the lungs indicated that the natural defence mechanisms of the lung against oxidative stress were sufficient to protect the tissues in group 2 against a weak oxidative stress which might be induced by V2O5. In groups 3 and 4, however, DNA modifications were noted, as were histopathological findings that may have been the result of incomplete particle clearance from the lung and sustained oxidative stress induced by the oxide. However, the contribution from V2O5 of a direct toxic effect upon the respiratory epithelium cannot be ruled out. Based on these findings a No-Observed-Adverse-Effect-Level (NOAEL) of 0.246 mg/m3 may be established under the conditions of this study.