Vanadium

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-10-19 to 2011-11-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This range-finding study was conducted according to the study protocol and its amendments, as well as all applicable IITRI Standard Operation Procedures (SOPs). The study is comparable to the OECD Technical Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study) with respect to the standards of a range-finding study, especially to test conditions, particle size distribution of aerosols, actual concentration measurements of the test substance, bronchoalveolar lavage, gross pathology and histopatholgy.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

This study was conducted to determine the potential toxicity of aerosolized vanadium trioxide powder to male and female Sprague-Dawley rats exposed to the test substance via nose-only inhalation. The rats were exposed at concentrations of 0, 0.0022, 0.022 and 0.25 mg/L air (analytical) for six hours per day, five days per week, over the course of two weeks (excluding weekend days; yielding a total of 10 exposures). Evaluated toxicological endpoints consisted of mortality/clinical observations, body weights, food consumption, pulmonary lavage parameters (cell numbers, differentials, total protein and lactate dehydrogenase), organ weights, gross necropsy and histopathology.

GLP compliance:

no

Remarks:

except for the analysis of vanadium trioxide in collection filters. This analysis was conducted under GLP.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Divanadium trioxide

EC Number:

215-230-9

EC Name:

Divanadium trioxide

Cas Number:

1314-34-7

Molecular formula:

O3V2

IUPAC Name:

Divanadium trioxide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Vanadium trioxide (Supplier: Stratcor, Inc., Hit Springs, AR)
- Physical state: black powder (milled test substance)
- Storage condition of test material: stored at room temperature. Each time the container of bulk test substance was opened, it was triple-purged with argon prior to closing.
- Two shipments of the test substance was received by the testing facility. The two shipments of test substance were combined and thoroughly mixed. The mixed batch of test substance was used to generate the inhalation atmospheres for the study.
The composition of the first shipment of the test substance (percentage levels of vanadium and ten specific impurities as specified by the
Sponsor) was determined by testing facility using inductively coupled plasma-mass spectrometry (ICP-MS). Purity of the test substance (% vanadium) was determined by the testing facility using colorimetric titration.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS - CRL:CD®(SD)IGS BR
- Source: Charles River Laboratories' Portage, MI, facility
- Age at study initiation: 55 days
- Weight at study initiation: males: 203 - 239 g; females: 184 - 215 g
- Fasting period: approximately 16-19 hours prior to necropsy
- Housing: during the quarantine period, animals of the same sex were double-housed in polycarbonate cages with hardwood bedding. After randomization, animals were single-housed in stainless steel cages suspended over absorbent paper cage boards.
- Diet (ad libitum, except during inhalation exposure): Certified Rodent Diet No. 5002 (PMI Nutrition International, Inc., Brentwood, MO)
- Water (ad libitum, except during inhalation exposure): City of Chicago water
- Acclimation period: 6 days
To condition the animals to placement and restraint in the nose-only holding tubes, the animals were placed in the holding tubes over three days prior to the start of exposure to the test substance according to the following schedule: one and a halfhours on Day -5; three hours on Day -2; and 4 hours and 30 minutes on Day -1.

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

ENVIRONMENTAL CONDITIONS
- Temperature: 20-23°C
- Relative humidity: 32-64%
- Air changes: minimumof 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Remarks on MMAD:

MMAD / GSD: The overall mean MMAD values and the GSD ranges were as follows:
0.0022 mg/L: MMAD = 1.92 ± 0.12 µm; GSD = 1.70 - 2.49
0.022 mg/L: MMAD = 2.02 ± 0.09 µm; GSD = 1.67 - 2.85
0.25 mg/L: MMAD = 2.25 ± 0.06 µm; GSD = 1.53 - 2.38

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus/ Method of holding animals in test chamber: each flow-past nose-only inhalation exposure chamber (Lab Products Inc., Seaford, DE) is comprised of 52 ports. The test atmosphere inlet and exhaust configurations provided a uniform and continuous stream of fresh test atmosphere to the animals undergoing exposure.
The animals were restrained in nose-only exposure animal holding tubes (CH Technologies, USA, Westwood, NJ). Each tube was placed in a predesignated port of the inhalation exposure chamber. Chamber ports were rotated for each exposure.

- System of generating particulates/aerosols: the test atmosphere was generated by aerosolization of the test substance using compressed air-operated Wright Dust Feeder Aerosol Generation systems (BGI Incorporated, Waltham, MA), which were positioned over each chamber. The test substance was weighed and packed into a dust reservoir using a 10 ton hydraulic shop press (TorinJacks, Ontario, CA), forming a cake. Each cake was compressed to 2 tons with the hydraulic shop press. A constant-speed rotating scraper separated a thin film of the test substance at the surface of the cake and delivered it into a dispersing unit, drawn in by aspiration and dispersed by a high-velocity air jet. The resulting test atmosphere entered a mixing plenum where it, when necessary, was diluted with breathable quality compressed air to achieve target concentration prior to introduction to the exposure chamber.
Exhaust from the exposure chambers was moved through a high efficiency particulate air (HEPA) filter by a ring compressor and exhausted outside the building. Inlet and exhaust flows to and from the chamber were controlled and continuously monitored by rotometers.

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

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

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

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

TEST ATMOSPHERE
- Brief description of analytical method used: the test atmosphere concentration in each exposure chamber was determined gravimetrically each exposure day by collecting test atmosphere samples on membrane filters placed in closed-face filter holders in the breathing zone of the animals. The gravimetric sampling train consisted of a preweighed 47 mm Teflon membrane filter (GE Water and Process Technologies, Westborough, MA) in series with a dry-gas meter connected to a constant flow vacuum pump. Samples were collected at a constant flow rate equal to the port flow of the delivery tube. The filter samples were weighed to determine the aerosol mass collected. The dry-gas meter measured the corresponding volume of chamber
air sampled and the weight-to-volume ratio was determined to obtain the aerosol mass concentration. Samples were collected at the following frequencies:
0 mg/L: 360 minutes (nominal sampling time); 1 sample/exposure
0.0022 mg/L: 360 minutes (nominal sampling time); 1 sample/exposure
0.022 mg/L: 120 minutes (nominal sampling time); 3 sample/exposure
0.25 mg/L: 30 minutes (nominal sampling time); 3 sample/exposure
One filter each from the 0.022 and 0.25 mg/L group Exposure/Study Day 1 exposures was analyzed by ICP-MS for determination of vanadium content and to confirm the gravimetric weight measurement. A filter from 0.0022 mg/L group was not analyzed by ICP-MS due to the small quantity of material collected on filters.
Test substance concentration values as determined by ICP-MS for one filter each from the 0.022 and 0.25 mg/L group Exposure/Study Day 1 exposures were 95% and 98% of gravimetric values for the two levels, respectively (please also refer to "Attached background material" below).
Aerosol concentration was monitored with a real-time aerosol sensor (model#pDR-1000AN, MIE, Inc. Bedford, MA). The sensors were employed as a realtime indicator of short term changes in aerosol concentration and were used in guiding laboratory personnel if concentration excursions were encountered.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Please refer to "Details on inhalation exposure" above.

Duration of treatment / exposure:

2 weeks (excluding weekend days; yielding a total of 10 exposures)

Frequency of treatment:

6 hours per day, 5 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.002 mg/L air (analytical)

Doses / concentrations 2

Dose / conc.:

0.022 mg/L air (analytical)

Doses / concentrations 3

Dose / conc.:

0.25 mg/L air (analytical)

No. of animals per sex per dose:

6 males / 6 females

Control animals:

yes, concurrent vehicle

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations: mortality, moribundity and general appearance

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

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

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

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

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

PULMONARY LAVAGE:
Bronchial alveolar lavage (BAL) was performed on Study Day 15 (after 10 exposures) on the left lobe of the lung of all study animals.
BAL fluid was analysed for the following: lactate dehydrogenase (LDH), protein analysis, cell counts (concentration, cells/lung, viable cells/lung and percent viable cells) and differential counts (segmented neutrophils, lymphocytes, monocytes/macrophages, eosinophils, and basophils)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Terminal fasted body weights were recorded on Study Day 15 (one day after the last exposure). The animals were euthanized by sodium pentobarbital followed by exsanguination from the abdominal aorta and were given a complete necropsy on Study Day 15.

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

HISTOPATHOLOGY: Yes
Tissue samples were trimmed, processed, embedded in paraffin, sectioned at approximately 5 µm, mounted on slides, and stained with hematoxylin
and eosin for the groups as follows:
- Air control group and 0.25 mg/L group males and females - bronchi, lung (right), bronchial lymph node, trachea, mediastinal lymph node, thymus and gross lesions
- 0.002 mg/L group and 0.02 mg/L group males and females - tissues identified as target tissues; e.g., lung, bronchial lymph node, mediastinal lymph node and thymus
Histopathological findings were recorded.

Statistics:

Descriptive statistics (mean and standard deviation) were calculated for data in the following categories: test atmosphere, body weight/body weight change,
food consumption, pulmonary lavage, and organ weight/organ-to-body weight ratios. The continuous data in the above categories, with the exception of test
atmosphere, were analyzed for statistical significance. A minimum significance level of p ≤ 0.05 was used for the comparisons.
If the data sets were normally distributed and of equal variance, statistical comparisons were conducted using one-way analysis ofvariance (ANOVA), with post hoc comparisons (if necessary) made using Dunnett's test. If normality and/or equal variance tests failed for a data set, statistical comparisons were conducted using nonparametric Kruskal-Wallis ANOVA followed (if necessary) by Dunn's test.
Body weight/body weight change, food consumption, the pulmonary lavage parameter ofLDH, and organ weight/organ-to-body weight ratios were compared
using ToxData® (version 2.1.E). The remaining pulmonary lavage parameters (cell numbers, differentials and total protein concentration) were compared using
SigmaStat® Software, version 2.03 (Systat Software Inc., Chicago, IL).
The differentials category ofbasophils (pulmonary lavage) was not analyzed for statistical significance because all results were "0" for all animals ofboth sexes in all groups.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No test-substance related mortality or clinical signs of systemic toxicity were observed.

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

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

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

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

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

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

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

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

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

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

Increases, although not statistically significant, also were noted in cell concentration in 0.0022 mg/L group males and females; cells/lung and viable cells/lung in 0.0022 mg/L group males; protein concentration in 0.0022 mg/L group females; and LDH in 0.0022 mg/L group females.
For most of the BAL parameters, there was an exposure concentration response for the low and mid exposure groups; however, the values for the high exposure group were lower than those for the mid group. This may have been the result of the inability to fully recover the infused lavage fluid (possibly due to the lung being filled with particulate vanadium, as evidenced microscopically by pigmentation of alveolar macrophages, thus preventing removal/collection of the cells filling the alveoli).
No eosinophils and basophils were detected in the BAL fluid of all animals of both sexes in all groups including controls.

Effect levels

1

Dose descriptor:

NOAEC

Effect level:

0.022 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

2

Dose descriptor:

LOAEC

Effect level:

0.25 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The no-observed-adverse-effect concentration (NOAEC) in this study is established at the exposure concentration of 0.02 mg/L based on decreased body weights at the high exposure level of 0.25 mg/L (LOAEC). The changes of BAL parameters, lung weights and lung histopathology seen at all exposure levels can be considered adaptive responses to the exposure to vanadium trioxide aerosols.