Divanadium pentaoxide

Carcinogenicity

Description of key information

Information on the carcinogenic potential is taken from an NTP study (k_NTP 2002) with exposure of male and female rats and mice over 2-years, and there is no evidence of systemic carcinogenicity in a study with V2O5. The marginal evidence for carcinogenicity in the animal lung of mice but not rats in the study with V2O5 is considered a substance specific local effect (see discussion). Human data reporting a carcinogenic potential do not exist. Classification for carcinogenicity should be examined once the needed data will be generated.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1997-01-06 (first exposure) to 1999-01-04/08 (necropsy date)

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

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 or test system 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.

Quality of the NTP study
The NTP carcinogenicity study was introduced in the CLH proposal and by the RAC opinion as a “well-conducted” study. However, there are points that may be considered, drawing the reliability of this study into question:
1. dose-range finding experiments were commissioned by NTP in two different labs (i.e. IITRI and BNL), using identical rat and mice strains but resulting in significantly different effects in the respiratory tract at the same exposure concentrations (e.g. bronchiolar hyperplasia was seen in all mice exposed to 1mg/m³ in the BNL cancer study already after 2 months but not in the IITRI study after 3 months exposure) – see also MacGregor, 2020. The most prominent difference between the two test setups were different aerosol generators. It is worth highlighting that differences in the dust generation methods also lead to significant differences in the acute inhalation toxicity studies reported by Anonymous (2011) and Leuschner (1994). This raises a concern that the effects seen in the chronic inhalation experiment run at BNL may be cause of the test material treatment than by the substance itself.
2. The formation of lung tumours in mice showed a flat, plateau-like dose response with increasing V2O5 concentrations is secondary to chronic inflammatory changes which developed shortly after initiating exposure and persisted throughout the study. The 16-day inhalation study in mice by Schuler et al. (2011) showed lung inflammation as well as increased cell proliferation at the same exposure levels. Thus, the interpretation of tumour data is confounded by the presence of long-term chronic inflammation.
3. Further, the NTP study shows shortcomings in the (i) test item characterisation, which was chemically analysed only 2 years before study start and (ii) the chemical identity of the test item was analysed only once in the inhalation chamber (see also MacGregor 2020, and Duffus, 2007).
4. Levels of vanadium in the blood of untreated control laboratory rodents are typically extremely low and usually below the limit of detection (Roberts et al., 2019; Schuler et al. 2011: < 0.013 µg/g). However, in appendix K to the NTP (2002) report (p304 & 307), the V blood levels in unexposed control mice are reported with 0.26 - 0.5 µg V/g blood. The tap water levels in the city in which the NTP study was conducted and also the NTP 2000 diet that the animals were fed during the study was checked for presence of vanadium contamination; in both cases, vanadium levels are well below 1 ppb, which practically excludes these sources as relevant contributors to the reported blood vanadium levels. Whereas the RAC opinion speculates about a possible analytical or calculation error, we believe this can be ruled out, because (i) the method validation report in the Annexes of the NTP study documents linearity of the analytical method in the required range and also reports LoD and LoQ (LoQ being 0.2 µg V/g blood) (ii) all blood vanadium levels in the NTP are above this LoQ and can therefore not be discounted (iii) NTP acknowledged that the V levels in control animals are far too high but also categorically rule out any analytical errors. They however offer no other explanation
Regarding the relevance of NTP blood vanadium levels, when comparing the vanadium blood levels of the three exposure groups to the NTPs own estimations of resulting blood V concentrations, the inhaled amounts contribute maximally to one third of the blood vanadium. Given that the animals were exposed whole body throughout, the average measured blood V concentrations above control amount to as much as approx. 500 µg V/L blood (highest exposure group). Since diet and water can be ruled out as relevant sources of Vanadium, this likely represents inadvertent ingestional uptake such as translocation from the respiratory to the GI tract as well as grooming etc.
According to the results of the deposition modelling of the mice in the NTP study using the MPPD mode, almost 50% of the inhaled material is translocated to the gastro-intestinal tract. By comparison with the recently published NTP sub-chronic drinking water rodent studies (Roberts et al. 2019) with two different Vanadium substances, such a blood level of Vanadium (500 ug/L blood) would be as much as 5-fold above the blood vanadium resulting from the highest drinking water exposure level of 100 µg V/L. This latter level however constituted the MTD, at which drinking water consumption was already substantially reduced. In consequence, the animals in the NTP study can be assumed to have been orally exposed to levels approx. 5-fold above the sub-chronic MTD; and any oral chronic MTD should not expected to be above the sub-chronic one.
The inhalation exposures in the NTP study are associated at the highest exposure level with blood vanadium levels well above the oral MTD for vanadium. Tumours resulting from this substantial systemic exposure were not observed. Additionally, vanadium pentaoxide only acts as such at the site of contact (such as lungs and GI tract), where its oxidation properties and the acidification during dissolution in aquatic media are likely the main cause of the inflammatory reactions. However, after dissolution vanadium pentaoxide no longer exists as such, but has instead transformed to vanadate in anionic form which no longer has these potent inflammatory properties. This is likely the reason for the complete absence of systemic tumours. In essence, the high vanadium blood levels in the NTP study should be considered a reflection of substantial inhalation and ingestional exposure, which is without systemic carcinogenicity.

All in all, the points raised above raises substantial issues as to the quality of the study and the reliability of the results.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

0.5 mg/m³

Study duration:

chronic

Species:

rat

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

There is no evidence of systemic carcinogenicity in a study with V2O5, which is a suitable surrogate regarding systemic effects. The marginal evidence for carcinogenicity in the animal lung of mice but not rats in the study with V2O5 is considered a substance-specific local effect.Because the data base is regarded as insufficient for the derivation of an Occupational Exposure Limit with respect to the endpoints carcinogenicity and genotoxicity by various committees and for these above reasons including the fact that human data reporting a carcinogenic potential do not exist, classification for carcinogenicity should be examined once the needed data will be generated.

Additional information

No carcinogenicity, no pneumoconiosis and no other signs indicative of allergic inflammation have been reported for workers manufacturing divanadium pentaoxide. 

 

Considerable thought has been given to the possible classification of divanadium pentaoxide as a carcinogen based upon the available scientific data. There is in fact no credible human epidemiological evidence for carcinogenicity, and we must thus rely upon the experimental studies and any in vitro mechanistic information that is available for an assessment. Workers (63) exposed at 0.1 to 3.9 mg V/m³(average 0.2-0.5 mg V/m³) measured as total dust for 11 years in a factory manufacturing divanadium pentaoxide did not have an increased prevalence of upper respiratory symptoms in the human case study by Kiviluoto et al (1979a,b, 1980, 1981 a, b). No cancer, no pneumoconiosis and no other signs indicative of allergic inflammation, including nasal catarrh, cough, phlegm, were observed in the exposed subjects working for 11 years under these occupational conditions. A statement on the preferential use of human data in risk assessments for human health is attached in section 7.5.

 

A two year carcinogenicity study (NTP 2001) is available, in which the substance V2O5 was administered to rats and mice via inhalation. The results of this study can be summarised as follows:

Rats: 

The incidences of alveolar/bronchiolar adenoma (including multiple) in males were 4/50, 8/49, 5/48, and 6/50 for the exposure concentrations of 0, 0.5, 1, and 2 mg/m³, respectively. Incidences of alveolar/bronchiolar carcinoma (including multiple) were 0/50, 3/49, 1/48, and 3/50, and of alveolar/bronchiolar adenoma and carcinoma (combined) 4/50, 10/49, 6/48, and 9/50 for the respective concentrations. In females, the incidences of alveolar/bronchiolar adenoma were 0/49, 3/49, 1/50, and 0/50, and those of alveolar/bronchiolar adenoma and carcinoma were 0/49, 3/49, 1/50, and 1/50 at the respective concentrations.

 

Initially, NTP (2002) stated that there were no statistically significant pairwise differences in the poly-3 test of lung tumour rates between treated and concurrent control group rats of either sex. The conclusion drawn by NTP that “some evidence of carcinogenic activity in male F344/N rats” and “equivocal evidence of carcinogenic activity in female F344/N rats” was based on comparisons of divanadium pentaoxide-treated group incidence rates with limited incidence lung cancer rate ranges for historical control groups from 9 (or 10, for female rats) previous carcinogenicity studies conducted with the NTP-2000 diet (as used in the V2O5 bioassay) and from the earlier NTP historical database using the NIH-07 diet (16 studies).

 

Later, NTP (2011) published a revised summary report of tumour incidence rates in control groups of F344/N rats fed the NTP-2000 diet in 25 carcinogenicity studies performed subsequent to the V2O5 study (NTP, 2002). Using these additional and more relevant NTP historical control data (NTP 2011) for supplementation of the NTP-2000 historical control data summarized in the V2O5 bioassay report (NTP 2002), Starr et al. (2012) re-evaluated the evidence regarding the carcinogenicity of divanadium pentaoxide in rats in relation to this larger and more relevant historical lung tumour database:

 

The re-analysis with the pooled historical control tumour incidence data from 34 NTP studies (all using the NTP-2000 diet) resulted in a widening of the background tumour incidence ranges reported in the V2O5 Technical Report 507 (NTP 2002). In particular, the historical control range for male rat alveolar/bronchiolar carcinoma increased from 0-2% (NTP TR 507) to 0-6%. Thus, the alveolar/bronchiolar carcinoma incidence rates in the low and high V2O5 concentration groups of male rats (3/49 and 3/50, respectively) were at the upper end but within the updated historical control range for this tumour, not three times above it, as concluded on the limited NTP TR 507 database. With respect to the incidence of alveolar/bronchiolar adenoma in the 0.5 mg/m³ V2O5 group of female rats (3/49), this incidence is within the updated historical control range for this tumour of 0-8%, not at the upper end of the range. The analyses also characterized the concurrent male rat control group from the V2O5 bioassay as possibly being an “outlier” relative to the expanded historical control database, at least in relation to lung tumour incidence. That would account for the fact that the unadjusted male alveolar/bronchiolar adenoma incidence rate (8/49) at 0.5 mg/m³ and the unadjusted male alveolar/bronchiolar adenoma or carcinoma incidence rates at 0.5 and 2 mg/m³ (10/49 and 9/50, respectively) exceed the upper bound of the appropriate historical control incidence rate ranges (0-12% and 0-14%, respectively) for these tumours.

 

Taken altogether, the use of historical control data from carcinogenicity studies performed subsequent to a given bioassay in evaluating the evidence regarding the carcinogenicity of a substance is argued for as follows (Starr et al. 2012):

 

None of the poly-3 adjusted pairwise comparisons of alveolar/bronchiolar tumour incidences in treated and concurrent control groups of male or female rats from the V2O5 study were statistically significant (NTP 2002).

Further, the carcinogenicity conclusions drawn by NTP for male and female F344/N rat alveolar/bronchiolar tumours in the V2O5 study (NTP 2002) as providing “some” and “equivocal” evidence of carcinogenic activity, respectively, are entirely based on the comparisons of the treated group tumour incidence rates in the V2O5 bioassay with tumour incidence rate ranges for earlier historical control groups, only a limited number of which were fed the same NTP-2000 diet. Importantly, NTP (2002) recognized that the additional inclusion and use of the earlier NTP-07 diet historical control database might be inappropriate because tumour incidence rates can be affected by diet.

 

The use of additional historical control data for tumour incidences from 25 NTP carcinogenicity studies with F344/N rats conducted subsequent to the V2O5 study, all of which employed the NTP-2000 diet (NTP 2011), resulted in an extended database for assessment of the historical control incidence rate ranges of alveolar/bronchiolar neoplasms.

 

Based on their comparative re-evaluation of the observed lung tumour incidences in rats with pooled historical control tumour incidence data from 34 NTP studies, Starr et al. (2012) came to the conclusion that there is “no” evidence of divanadium pentaoxide carcinogenicity in male or female F344/N rats, which is in clear contradiction to the statement in the CLH Report with respect to lung tumour formation based on the NTP (2002) conclusions.

 

 

Mice:

 

In male mice, the incidences of alveolar/bronchiolar adenoma (including multiple) were 13/50, 16/50, 26/50, and 15/50 for the exposure concentrations of 0, 1, 2 and 4 mg/m³, respectively. Incidences of alveolar/bronchiolar carcinoma (including multiple) were 12/50, 29/50, 30/50, and 35/50. Alveolar/bronchiolar adenoma and carcinoma (combined) occurred with statistically significant increased incidences in all exposed males (22/50, 42/50, 43/50, 43/50). In female mice, the incidences of alveolar/bronchiolar adenoma (including multiple) were 1/50, 17/50, 23/50, and 19/50, and those of alveolar/bronchiolar carcinoma (including multiple) were 0/50, 23/50, 18/50, and 22/50. Alveolar/bronchiolar adenoma and carcinoma (combined) occurred with incidences of 1/50, 32/50, 35/50, and 32/50.

 

The incidences of alveolar/bronchiolar carcinoma and alveolar/bronchiolar adenoma or carcinoma (combined) were significantly increased at a maximum level across all three tested concentrations in exposed groups of male and female mice. The incidences of alveolar/bronchiolar adenoma were significantly increased in males exposed to 2 mg/m³ and in all groups of exposed females (NTP, 2002).

 

The significantly increased rates of tumours (alveolar/bronchiolar carcinoma) seen in the lung of all mice (males and females) exposed to 1, 2, or 4 mg/m³ as compared with those in the chamber controls amounted to 58%, 60%, 70% vs. 24% for males and 46%, 36%, 44% vs. 0% for females, respectively, thus exceeding the historical ranges for controls (all routes) given the NTP-2000 diet.

 

NTP concluded that there was clear evidence of carcinogenic activity of divanadium pentaoxide in male and female B6C3F1 mice (NTP, 2002). However, a flat plateau-like dose response for lung tumour formation in mice, especially in males, was seen with increasing V2O5 concentrations. These tumour rates do not appear to be dose-related over the narrow range of V2O5 concentrations that were tested. Poly-3-adjusted Cochran-Armitage trend test analyses without inclusion of the lung tumour incidence data from control group mice confirmed the absence of any significant dose-related effect on mouse lung tumour incidence in the study groups exposed to 1, 2 or 4 mg/m³ V2O5 (Starr & MacGregor, 2014).

 

NTP chronic study designs are comprehensive and include evaluation of an extensive list of tissues from all animals at all levels tested. Following these extensive tissue microscopic evaluations, only the respiratory tract was found to be affected in this 2-year chronic divanadium pentaoxide inhalation study (NTP, 2002). There was evidence of carcinogenic activity of V2O5 in male and female B6C3F1 mice based on increased incidences of alveolar/bronchiolar neoplasms. Exposure to divanadium pentaoxide 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. No other tissues showed macroscopic or microscopic treatment-related lesions.

 

Importantly, no increase in tumours was seen at any other target site in mice as well as in rats. This is noteworthy because vanadium levels were measured in blood, indicating internal systemic exposure and that the form of vanadium circulating would appear not to be toxic at the levels measured (NTP, 2002).

 

Lung tumour formation in mice, especially in males, showed a flat, plateau-like dose response with increasing V2O5 concentrations (NTP, 2002). Further, the absence of any significant dose-related effect on mouse lung tumour incidence in the study groups exposed to concentrations of 1, 2 or 4 mg/m³ V2O5 was confirmed by poly-3-adjusted Cochran-Armitage trend test analyses without inclusion of the lung tumour incidence data from control group mice (Starr & MacGregor, 2014). Thus, the tumour response in B6C3F1 mice exposed to 1-4 mg/m³ V2O5 is considered to be due, at least in part, to other factors than V2O5 airborne concentration or vanadium lung burden. The incidences of alveolar epithelial hyperplasia and bronchiolar epithelial hyperplasia were both significantly increased in a dose-dependent manner. However, chronic inflammation and alveolar histiocytic infiltration were observed in lungs of nearly all exposed mice at the end of the NTP study (NTP, 2002), e.g., male mice showed nearly maximum incidences of 42/50, 45/50, 47/50, and 36/50, 45/50, 49/50, respectively, at exposure levels of 1, 2, and 4 mg/m³. Based on these clear inflammatory responses, it was suggested that all of the tested V2O5 concentrations were excessively high, particularly for the low and intermediate tested dose groups, thus resulting in saturation of certain inflammatory mechanisms leading to toxicity.

 

Findings of the so-called 16-day special study in female mice (NTP 2002, cf. MacGregor et al., unpublished) indicated the occurrence of lung inflammation by Day 13 at exposure levels of 2 mg/m³ and above. Because the 16-day inhalation study in female B6C3F1 mice by Schuler et al. (2011) showed lung inflammation as well as increased cell proliferation at the same exposure levels, the interpretation of tumour data is confounded by the presence of long-term chronic inflammation, presumably over the whole duration of the two-year exposure duration.

 

The formation of lung tumours is secondary to chronic inflammatory changes which developed shortly after initiating exposure and persisted throughout the study. Similar to mice, rats had comparable vanadium lung burdens and also showed similar increased lung inflammation and epithelial hyperplasia in response to V2O5 exposure for two years, but lung tumorigenicity was not substantiated, indicating a species difference in response to this persistent chronic inflammation.

 

In a tumour initiation/promotion study in several strains of male mice (Rondini et al., 2010), divanadium pentaoxide administered repeatedly via oropharyngeal aspiration did not initiate lung tumours, but increased tumour multiplicity initiated by methylcholanthrene in A/J and BALB/cJ mice, but not in C57BL/6J mice. Hence, V2O5 operated as a tumour promoter in A/J and BALB/cJ mice. These sensitive mice were also found to be more susceptible to V2O5-induced pulmonary inflammation.

 

The genotoxicity experiments (S. typhimurium gene mutations and in vivo mouse peripheral blood micronucleus assay) included in the NTP (2002) study showed negative results for mutagenic effects. There is also a substantial weight of evidence from other in vivo and in vitro investigations (Assem & Levy, 2009) that the mode of action underlying the lung tumours in mice in the NTP 2002 study is by non-DNA reactive mechanism(s). The study in female B6C3F1 mice by Schuler et al. (2011) and the initiation-promotion study by Rondini et al. (2010) provide further evidence of a non-genotoxic mechanism for tumour induction by divanadium pentaoxide. Whereas some vanadium compounds have been shown to produce a range of chromosome damages, guideline-conforming, state-of-the-art in vitro gene mutation studies performed with all three valency states of vanadium have unequivocally demonstrated an absence of such effects, thus ruling out direct DNA interactions (Lloyd, 2010).

 

The lack of significant induction of cII mutant frequencies in the lungs of male transgenic Big Blue (BB) mice exposed to 1 mg/m³ V2O5 (tumorigenic concentration) by inhalation for up to 8 weeks suggests that divanadium pentaoxide is unlikely to act via a mutagenic mode of action (Manjanatha et al. 2015). Further, the lack of significant changes in levels of Kras codon 12 mutations (GGT→GAT or GGT→GTT) following exposure of male BB mice at V2O5 concentrations of 1 mg/m³ for 8 weeks supports the idea that the accumulation of additional Kras mutants is not an early event in mouse lung carcinogenesis, and/or that the proliferative advantage of Kras mutant clones requires either longer expression times or larger cumulative V2O5 exposures (Banda et al. 2015). Furthermore, the data do not provide support either for any direct genotoxic effect of V2O5 on Kras in the context of the exposure conditions used, or for any early amplification of pre-existing mutation as being involved in the genesis of V2O5-induced mouse lung tumours.

 

Further evidence that genotoxicity is not a driving force in lung tumour formation by V2O5 comes from the study of biological perturbations following 90-day V2O5 exposure at tumorigenic levels by Black et al. (2015). The study assessed if any of these perturbations were consistent with genotoxicity or oxidative stress and compared V2O5 responses with those of 13 other lung tumorigens and non-tumorigens. Differential gene expression varied greatly among the compounds. V2O5 had 1,026 differentially expressed genes, 483 of which were unique to V2O5. Functional ontology enrichment indicated several possible effects on lipid metabolism as well as ontology categories associated with inflammation. These functional ontology results are consistent with evidence of epithelial hyperplasia, degeneration and inflammation in mice, but were not indicative of processes traditionally related to tumor initiation. There was not any evidence for enrichment of pathways associated with changes in cell cycle/proliferation, DNA-damage, or oxidative stress related pathways with the V2O5 differentially expressed genes. Given that divanadium pentaoxide in contact with aqueous media yields a strongly acidic pH (Klawonn, 2010), due to chronic inflammation promoted by this pH effect divanadium pentaoxide may act through an increase in inflammatory-related oxidative stress. 

In summary, the in vivo genotoxicity studies in mice following inhalation exposure do not support a genotoxic (DNA-reactive) mode of action for divanadium pentaoxide.

 

Quality of the NTP study

The NTP carcinogenicity study was introduced in the CLH proposal and by the RAC opinion as a “well-conducted” study. However, there are points that may be considered, drawing the reliability of this study into question:

1. dose-range finding experiments were commissioned by NTP in two different labs (i.e. IITRI and BNL), using identical rat and mice strains but resulting in significantly different effects in the respiratory tract at the same exposure concentrations (e.g. bronchiolar hyperplasia was seen in all mice exposed to 1mg/m³ in the BNL cancer study already after 2 months but not in the IITRI study after 3 months exposure) – see also MacGregor, 2020. The most prominent difference between the two test setups were different aerosol generators. It is worth highlighting that differences in the dust generation methods also lead to significant differences in the acute inhalation toxicity studies reported by Anonymous (2011) and Leuschner (1994). This raises a concern that the effects seen in the chronic inhalation experiment run at BNL may be cause of the test material treatment than by the substance itself.

2. The formation of lung tumours in mice showed a flat, plateau-like dose response with increasing V2O5 concentrations is secondary to chronic inflammatory changes which developed shortly after initiating exposure and persisted throughout the study. The 16-day inhalation study in mice by Schuler et al. (2011) showed lung inflammation as well as increased cell proliferation at the same exposure levels. Thus, the interpretation of tumour data is confounded by the presence of long-term chronic inflammation.

3. Further, the NTP study shows shortcomings in the (i) test item characterisation, which was chemically analysed only 2 years before study start and (ii) the chemical identity of the test item was analysed only once in the inhalation chamber (see also MacGregor 2020, and Duffus, 2007).

4. Levels of vanadium in the blood of untreated control laboratory rodents are typically extremely low and usually below the limit of detection (Roberts et al., 2019; Schuler et al. 2011: < 0.013 µg/g). However, in appendix K to the NTP (2002) report (p304 & 307), the V blood levels in unexposed control mice are reported with 0.26 - 0.5 µg V/g blood. The tap water levels in the city in which the NTP study was conducted and also the NTP 2000 diet that the animals were fed during the study was checked for presence of vanadium contamination; in both cases, vanadium levels are well below 1 ppb, which practically excludes these sources as relevant contributors to the reported blood vanadium levels. Whereas the RAC opinion speculates about a possible analytical or calculation error, we believe this can be ruled out, because (i) the method validation report in the Annexes of the NTP study documents linearity of the analytical method in the required range and also reports LoD and LoQ (LoQ being 0.2 µg V/g blood) (ii) all blood vanadium levels in the NTP are above this LoQ and can therefore not be discounted (iii) NTP acknowledged that the V levels in control animals are far too high but also categorically rule out any analytical errors. They however offer no other explanation

Regarding the relevance of NTP blood vanadium levels, when comparing the vanadium blood levels of the three exposure groups to the NTPs own estimations of resulting blood V concentrations, the inhaled amounts contribute maximally to one third of the blood vanadium. Given that the animals were exposed whole body throughout, the average measured blood V concentrations above control amount to as much as approx. 500 µg V/L blood (highest exposure group). Since diet and water can be ruled out as relevant sources of Vanadium, this likely represents inadvertent ingestional uptake such as translocation from the respiratory to the GI tract as well as grooming etc.

According to the results of the deposition modelling of the mice in the NTP study using the MPPD mode, almost 50% of the inhaled material is translocated to the gastro-intestinal tract. By comparison with the recently published NTP sub-chronic drinking water rodent studies (Roberts et al. 2019) with two different Vanadium substances, such a blood level of Vanadium (500 ug/L blood) would be as much as 5-fold above the blood vanadium resulting from the highest drinking water exposure level of 100 µg V/L. This latter level however constituted the MTD, at which drinking water consumption was already substantially reduced. In consequence, the animals in the NTP study can be assumed to have been orally exposed to levels approx. 5-fold above the sub-chronic MTD; and any oral chronic MTD should not expected to be above the sub-chronic one.

The inhalation exposures in the NTP study are associated at the highest exposure level with blood vanadium levels well above the oral MTD for vanadium. Tumours resulting from this substantial systemic exposure were not observed. Additionally, vanadium pentaoxide only acts as such at the site of contact (such as lungs and GI tract), where its oxidation properties and the acidification during dissolution in aquatic media are likely the main cause of the inflammatory reactions. However, after dissolution vanadium pentaoxide no longer exists as such, but has instead transformed to vanadate in anionic form which no longer has these potent inflammatory properties. This is likely the reason for the complete absence of systemic tumours. In essence, the high vanadium blood levels in the NTP study should be considered a reflection of substantial inhalation and ingestional exposure, which is without systemic carcinogenicity.

 

All in all, the points raised above raises substantial issues as to the quality of the study and the reliability of the results.

 

 

SCOEL evaluation

An evaluation of the data base for divanadium pentaoxide by the Scientific Committee on Occupational Exposure Limits (SCOEL, 2004) concluded that considering the available genotoxicity data on divanadium pentaoxide and other vanadium compounds it is not possible to clearly identify a threshold level below which there is no concern. Regarding the carcinogenic potential it was concluded that divanadium pentaoxide was found to be carcinogenic in rats and mice, but the biological mechanism underlying the initiation and promotion of pulmonary disease and lung cancer induced by vanadium pentoxide is not understood. In consequence, a health based occupational exposure limit (OEL) was not derived by SCOEL. In order to gain more insight into the mechanism of the carcinogenic potential, a 16-day inhalation toxicity study was conducted in female mice with the evaluation of specific endpoints (Schuler, 2010). However, the results of the first study suggest that further investigations are necessary to identify mouse-rat differences that might suggest a mode of action, to investigate the lack of effects in a comet assay seen in this in vivo study in a further in-vitro test on BAL/pulmonary cells, and to investigate the 8-oxodGua-specific lesions observed with respect to induction or repair inhibition.

 

Because the data base is regarded as insufficient for the derivation of an Occupational Exposure Limit with respect to the endpoints carcinogenicity and genotoxicity by various committees and for these above reasons including the fact that human data reporting a carcinogenic potential do not exist, classification for carcinogenicity should be examined once the needed data will be generated.

 

 

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

(i) chronic toxicity,

(ii) carcinogenicity, and 

(iii) multi-generation reproductive toxicity

is planned.

 

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

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

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

- 90-d oral toxicity studies (dosed feed: NaVO3; dosed water: VOSO4) with Harlan Sprague-Dawley rats and B6C3F1/N (dose: rats and mice: 0, 31.3, 62.5, 125, 250, or 500 ppm – ongoing, interim results reported under repeated dose toxicity, oral

- Organ systems toxicity, i.e. 28-d immunotoxicity study of NaVO3 (dosed-water) with female B6C3F1/N mice (dose: 0, 31.3, 62.5, 125, 250, or 500 ppm) - ongoing

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

 

It can reasonably be anticipated that these studies will be of high quality and relevance, and thus will serve as a more robust basis than the current data base with all its shortcomings. In addition, repeated-dose inhalation toxicity studies (14, 28, and 90 days) with various vanadium substances are planned within the Vanadium Safety Readiness Safety Program. These studies will address issues for which to date equivocal or no data at all exist. Further information on these studies can be found in section 7.5. Only upon availability of the results from these studies, it will be possible to render a more meaningful decision on whether or not testing for carcinogenicity is required. Therefore, for the time being this data requirement should be waived in consideration of animal welfare.