Vanadium, oxalate complexes

Endpoint summary

Description of key information

A comprehensive literature search was recently conducted for the vanadium category substances, to source relevant information for the hazard and risk assessment. For the group of the soluble vanadium substances, a limited number of studies is available and the different experimental approaches lead to a variety of endpoints measured.

Of the limited effects noted following oral exposure with soluble vanadium substances, it appears most likely that effects on haematological parameters are the most consistently reported among a number of investigators (Mountain et al 1953, Zaporowska et al. 1993, Scibior et al 2006, Scibior, 2005, NTP, 2002). Altogether, haematological effects have been found with a variety of different vanadium compounds including sodium metavanadate, vanadium pentoxide, and ammonium metavanadate supporting the use of this endpoint for risk assessment purposes.

 

Information on repeated dose toxicity following inhalation exposure to V2O5 is available in a NTP study (k_NTP 2002) with exposure of male and female rats and mice to V2O5 over 16-days, 3-months and 2-years. Pulmonary reactivity to vanadium pentoxide was also investigated following subchronic inhalation exposure in a non-human primate animal model. The rationale for read-across to vanadium, oxalate complexes is summarised below (see discussion).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Dose descriptor:

LOAEL

9.15 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Several supportive studies exist.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: inhalation

Type of information:

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

Adequacy of study:

supporting study

Study period:

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well reported study. Test procedure in accordance with generally accepted scientific standards and described in sufficient detail.

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:

Groups of 50 male and 50 female F344/N rats (approx. 6-7 weeks old) were exposed to V2O5 aerosols at concentrations of 0, 0.5, 1 or 2 mg/m3 by inhalation, 6 hours/d, 5 d/wk, for 104 weeks. The body weight was controlled initially and body weight and clinical finding were recorded every 4 weeks (until week 89) and every 2 weeks from week 92 on. Animals were observed twice daily. Necropsy was performed at study end.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

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

IN-LIFE DATES: From: 1997-01-06 (first exposure) to 1999-01-4/8 (necropsy date)
- No further information on test material was stated.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: MMAD = 1.0-1.3 µm, GSD = 2.3-2.8

Details on inhalation exposure:

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

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

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- During all studies, chamber aerosol concentrations were monitored with real-time aerosol monitors (RAMs) that used a pulsed-light-emitting diode in combination with a silicon detector to sense light scattered over a forward angular range of 45° to 95° by particles traversing the sensing volume. The instruments respond to particles 0.1 to 20 μm in diameter.
- For the 16-day special studies and the 2-year studies, the sampling system consisted of a valve that multiplexed each RAM to two or three exposure chambers and to a HEPA filter and/or the control chamber or room; selection of sampling streams and data acquisition from each RAM was remotely controlled by a computer. Equations for calibration curves were stored in the computers and were used to convert the measured voltages to exposure concentrations.
- Each RAM was calibrated daily during the 16-day and 3-month studies by correlating the measured voltage with vanadium pentoxide concentrations determined by gravimetric analysis of glass fiber filters and one to two times per week during the 2-year studies by ICP/AES or ICP/mass spectrometry analysis of Pallflex® TX40H120WW glass fiber filters.

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

6 hours per day, 5 days per week

Remarks:

Doses / Concentrations:
0.5 mg/m³ V2O5
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
1 mg/m³ V2O5
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
2 mg/m³ V2O5
Basis:
nominal conc.

No. of animals per sex per dose:

core study: groups of 50 male and 50 female rats
tissue burden analyisis: 40 female rats per exposed group; separate control groups of 15 female rats were used as chamber controls

Control animals:

yes

Details on study design:

- Dose selection rationale: based on the incidences and severities of respiratory lesions and increased lung weights in male and female rats, concentrations of 4 mg/m3 or greater were considered to be too high for use in a 2-year study. The exposure concentrations selected for the 2-year inhalation study in rats were 0.5, 1, and 2 mg/m3.
- Rationale for animal assignment (if not random): randomly into groups of approximately equal initial mean body weights.
- No further information on test material was stated.

Positive control:

not stated

Observations and examinations performed and frequency:

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

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

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

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

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

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

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

Sacrifice and pathology:

Method of sacrifice: CO2 asphyxiation.

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

HISTOPATHOLOGY:
- Complete histopathology was performed on all core study animals. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gallbladder (mice only), heart and aorta, large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum, and ileum), kidney, larynx, liver, lung and mainstem bronchi, lymph nodes (mandibular, mediastinal, mesenteric, and bronchial), mammary gland (except male mice), nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea,
urinary bladder and uterus.

Other examinations:

Five male and five female rats were randomly selected for parasite evaluation and gross observation of disease.

Statistics:

Survival Analyses:
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. Animals found dead of other than natural causes were censored from the survival analyses; animals dying from natural causes were not censored. Statistical analyses for possible doserelated effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.

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

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

only marginally less for the 2 mg/m3–exposed females

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

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.

Details on results:

CLINICAL SIGNS AND MORTALITY
- Survival of the animals were similar to the controls

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

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

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

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

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

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

HISTOPATHOLOGY: NEOPLASTIC (if applicable)

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

LUNG:
- Effects in males: alveolar/ bronchiolar adenoma (4/50, 8/49, 5/48, 6/50); alveolar/bronchiolar carcinoma (0/50, 3/49, 1/48, 3/50); alveolar/ bronchiolar adenoma or carcinoma (4/50, 10/49, 6/48, 9/50)
- Effects in females: none (equivocal findings: alveolar/bronchiolar adenoma (0/49, 3/49, 1/50, 0/50); alveolar/bronchiolar adenoma or carcinoma (0/49, 3/49, 1/50, 1/50)
- Alveolar/bronchiolar neoplasms with incidences often exceeding the historical control ranges, 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; incidence in the 0.5 mg/m3 group was at the upper end of historical ranges. 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.

Dose descriptor:

LOAEC

Remarks:

local

Effect level:

0.5 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: non-neoplastic changes (epithelial hyperplasia, squamous metaplasia, chronic inflammation, degeneration) in the respiratory system (lung, larynx, and nose) of male and female rats

Critical effects observed:

not specified

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

0.85 mg/m³

Study duration:

chronic

Species:

rat

Quality of whole database:

Findings in rat model may not be relevant for humans. Further, inhalation may not be a relevant route of exposure for vanadium, oxalate complexes (solution).

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Initial comment on grouping and read across – oral

The endpoint repeated dose toxicity via the oral route of the vanadium category substance is not addressed by substance-specific information but rather by read-across of data available for soluble tri-, tetra- and pentavalent vanadium substances as well as for insoluble vanadium substances with zero valency, such as vanadium carbide.

All vanadium substances within the read-across concept represent inorganic substances, including salts or oxides and form the vanadium category. For the substances of the inorganic vanadium substances category, it is assumed that the in vivo bioavailability of the vanadium varies in a predictable manner, which is dependent on the in vitro bioaccessibility of the respective vanadium substance, i.e. all members of the category liberate vanadium ions in aqueous media at different rates, inter alia depending on the chemical structure. Thus, this concept is based on the chemistry / composition of all substances and on experimental studies for (i) water solubility and (ii) in-vitro bioaccessibility: assessment of the solubility and speciation of vanadium substances in five different artificial physiological fluids. Robust summaries for these studies are provided in each registration dossier and a detailed evaluation of the relevance, reliability and adequacy of each study is presented in the individual study records. Based on the in vitro bioaccessibility and the available bridging studies, two read-across groups are defined for the systemic effects following oral exposure, i.e. (i) soluble vanadium substances and (ii) poorly soluble vanadium substances. This endpoint summary addresses the hazard data for the soluble read-across group. Further details on the read-across concept are presented in the report attached to IUCLID section 13.

Therefore, the remaining text in this chapter is generic for all vanadium substances of the soluble read-across group and has not been adapted on a substance-specific basis.

 

Oral - animal data:

A number of studies are available reporting findings observed mainly in rats but also in mice orally exposed to soluble tetra- and pentavalent vanadium substances. However, the studies applied different experimental designs and were targeted at investigating specific endpoints. Thus, the different experimental approaches led to results on a variety of endpoints, which is why the weight of evidence approach is applied.

Based on the available data, the following adverse effects were identified: body weight reduction, haematological effects and reduction of antioxidants in tissues and body fluids. These effects are the most consistently reported among a number of investigators (Mountain et al. 1953, Dai et al. 1985, Roberts et al. 2015 and 2019, Zaporowska et al. 1993, Scibior et al. 2005, 2006, 2012).

 

A detailed review of the oral repeated dose toxicity studies in animals is presented below.

 

Mountain et al. 1953: Male rats were treated with vanadium pentoxide for up to 103 days. The experimental setup was split into three phases: 1. animals were exposed to 0, 100 and 150 ppm V for 103 days; 2. animals were exposed to 0, 500 and 1000 ppm V and one group-paired fed control for the 500 ppm group for 75 days; 3. animals were exposed to 0 and 500 ppm V and an additional group was fed with a diet containing 500 ppm V and 1.6% methionine for 63 days. In all three setups body weight gain and food intake as well as amount of ingested vanadium were recorded. In the first two setups of the study the cysteine content of hair was determined three times (1. setup: day 0, 60 and 103; 2. setup: day 0, 36 and 75). Additionally, in the first setup, red cell count and haemoglobin was determined before study initiation and at study termination. In the third setup liver to body weight ratio was recorded. According to the author, hair cysteine content decreased dose-dependently in animals exposed to 100, 150, 500 and 1000 ppm vanadium. The food intake was not significantly affected in animals exposed to vanadium. However, animals exposed to 500 and 1000 ppm vanadium showed significantly decreased body weight gains after 75 days (500 and 1000 ppm V) and 63 days (500 ppm V). Animals exposed to 100 and 150 ppm vanadium for 103 days showed slightly decreased red cell counts and haemoglobin levels. No effects on liver to body weight ratios were observed in animals exposed to 500 ppm vanadium for 63 days.  Since the effects on red cell count and haemoglobin in animals exposed to 100 and 150 ppm for 103 days were mild and within historical control ranges of this rat strain and age, the effects were considered to be negligible. Further, cysteine content of rat hair was slightly reduced but the adversity of this effect remains questionable. Since body weight and food consumption in animals exposed to 100 and 150 ppm V were normal and no further adverse effects were observed, mildly decreased values of cysteine in hair is considered not adverse. Consequently, 150 ppm V (equivalent to 4.58 mg V/kg bw/day) represents the NOEL of this study, based on the reduced body weight gain in animals exposed to 500 and 1000 ppm vanadium. This study was rated as RL 3 due to significant methodological deficiencies.

 

Dai et al. 1994: Four groups of non-diabetic male rats received different concentrations of vanadyl sulfate (VOSO4) in drinking water for 52 weeks. The low dose group received 500 mg VOSO4/L in water for 52 weeks. The mid dose group received 500 mg/L for 1 week followed by 750 mg VOSO4/L in water for 51 weeks. The high dose group received 500 and 750 mg/L for 1 week each followed by 1250 mg VOSO4/L in water for further 50 weeks. Three recovery animals of each group were followed for further 16 weeks after cessation of VOSO4 treatment. Treatment of male rats with different dose levels of vanadyl sulfate in drinking water corresponding to 34, 54 and 90 mg/kg bw/day over 52 weeks did not indicate severe signs of systemic toxicity under the conditions of this study. Body weights were dose-dependently reduced in treatment groups compared to controls, occasionally reaching statistical significance in the low and mid dose groups and at most time points in the high dose group. At study termination (week 52) significant difference in body weight (>10%) compared to control animals was observed in high dose animals only. Body weights of low and mid dose animals were lower (<10%) but not significantly different and thus considered not biologically relevant. Based on significant and biologically relevant effects on body weight in high dose animals, the mid dose level of 54 mg/kg bw/day represents a NOAEL. This study was rated as RL 2, comparable to guideline study with acceptable restrictions.

 

Domingo et al. 1985: Sodium metavanadate in drinking water was given to four groups, each consisting of 10 male Sprague-Dawley rats, at concentrations of 0, 5, 10 and 50 ppm. Liver and renal function parameters were determined in blood at the end of exposure period and organ weights were taken (liver, kidneys, heart, spleen, lung). Heart, liver, lungs, kidneys, spleen, stomach, small and large intestine were histopathologically examined in three rats of each group. Oral administration of NaVO3 via drinking water to groups of male rats over 3 months at concentrations of 0, 5, 10 and 50 ppm caused mild, dose-dependent lesions in kidneys, lungs and spleen with the highest incidence in the 50 ppm group, and increased plasma concentrations of protein, urea and uric acid in the high dose group. Thus, the highest dose level (7.57 mg/kg bw/d NaVO3) represents a clear LOAEL, and the mid dose level (1.51 mg/kg bw/d NaVO3) represents a NOAEL. This study was rated as RL 3 due to significant methodological deficiencies.

 

Zaporowska et al. 1993: Male and female Wistar rats were orally exposed to ammonium metavanadate (0, 10 or 50 mg V/L ) in drinking water for 4 weeks. Body weight gain, food and fluid uptake as well as various haematological and biochemical parameters were determined. Effects on palatability of vanadium containing fluids was observed at 50 mg V/L (significant in males but not in females). Mild and not dose-dependent effects were observed on body weight gain. Additionally, slight but significant effects on erythrocytes, reticulocytes and haemoglobin as well as L-ascorbic acid level in plasma and erythrocytes were observed in both dose groups. As no dose dependency was observed, mild effects on body weight gain were considered negligible. Significant effects on haematological parameters were considered to be mild and within historical control ranges of this rat strain and age (discussed below). Effects on L-ascorbic acid level in erythrocytes and plasma were also not considered relevant since the adversity of this effect is unclear (discussed below) and no further adverse effect was observed. Based on this, the NOEL of this study is considered to be 50 mg V/L drinking water, which is equivalent to 4.93 and 6.65 mg V/kg bw/day in males and females, respectively. This study is well reported, however, no guideline was followed and only limited parameters were analysed. Erythrocytes and haemoglobin and reticulocytes were slightly but significantly changed in both dose groups and sexes. These changes were not considered to be adverse as they were minor compared to control and within historical control data of this rat strain and age (e.g. Kort, M. et al. (2020) and Giknis, M. & Clifford, C.B. (2008)). Apart from the above-mentioned minor effects on some haematological parameters, the level of L-ascorbic acid was significantly decreased in plasma of low and high dose males but not in females. The same author reported one year later (please refer to Zaporowska 1994) that male and female rats exposed to doses of up to 0.30 mg V/ml showed clearly reduced L-ascorbic acid values in liver, kidney, adrenals and spleen. Thus, exposure to ammonium metavanadate seems to correlate with decreased L-ascorbic acid values in different tissues and body fluids. However, as no systemic toxicity or any other effect was observed, it remains unclear whether mild to moderate L-ascorbic acid depression is or results in any adverse effects. In a publication of Chan & Reade (1996) Wistar Shionogi rats, unable to synthesize L-ascorbic acid, were supplemented with different doses of L-ascorbic acid to determine the L-ascorbic acid requirements in Wistar rats. After 26 weeks, all animals survived and showed no clinical signs of scurvy. The average weekly body weight gain was normal. A severe L-ascorbic acid deficiency would include perinasal and peri-and intra-oral haemorrhage, joint or intramuscular haemorrhage, weakened or fractured hind limbs, delayed wound healing and a failure to thrive (Clemetson, 1989). As none of these clinical signs were observed in studies reported by Zaporowska (1993, 1994) or Chan & Reade (1996), it is assumed that this mild to moderate L-ascorbic acid depression observed in this study is not an adverse effect. It has been demonstrated that the toxicity of vanadium increases with its valency. Thus, compounds containing pentavalent V, such as ammonium metavanadate, are most poisonous. It is known that pentavalent V enters cells through anion channels, i.e. phosphate or sulfate channels. In cells, pentavalent V is reduced to divalent VO by some reducing compounds such as L-ascorbic acid and thiol-containing cysteine. Thus, the reported reduction of L-ascorbic acid in plasma and erythrocytes and also in several tissues (Zaporowska, 1994) is most likely the result of an enhanced consumption/reduction activity of this compound. However, in contrast to humans, rodents are able to synthesize L-ascorbic acid. Thus, it can be assumed that this reduction of L-ascorbic acid will induce re-synthesis and reduction of L-ascorbic acid is an adaptive but not an adverse effect. Apart from that, it is also noteworthy that humans are not able to synthesize L-ascorbic acid. Based on this, it is assumed that the protective reduction of pentavalent V to divalent VO is performed by another reducing substance and thus, it remains questionable whether a reduction of L-ascorbic acid in rats is relevant for humans. This study was rated as RL 3 due to significant methodological deficiencies.

 

 

The following studies reported similar effects on haematological parameters and body weight and also a few other signs of systemic toxicity, but were regarded as supportive, as several shortcomings in study design and reporting led to limited relevance in risk assessment.  

 

Scibior et al. (2012) and Scibior et al. (2018): A group of 16 male Wistar rats received sodium metavanadate (13.03 mg vanadium/kg bw/day) in drinking water for 12 weeks. The following parameters were investigated: clinical signs, mortality, physical appearance, motor behaviour, food/water consumption, haematology, clinical chemistry, and urinalysis (iron concentration only). Furthermore, atomic absorption measurements of vanadium, magnesium, iron, copper and zinc were conducted in selected biological material (liver, spleen, faeces, red blood cells, and urine). Lastly, femoral diaphysis were prepared and analysed for oxidative stress markers (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, total glutathione, glutathione disulfide, lipid peroxidation) and L-ascorbic acid (also in the liver) as well as metal content (vanadium, magnesium, calcium, iron, copper and zinc).

 

Scibior et al. (2012): After the administration of 13.03 mg/kg bw/day of sodium metavanadate in drinking water some rats had gastrointestinal disturbances. Loose stool/diarrhoea was observed. No mortality was observed in the study. Furthermore, the treatment with sodium metavanadate led i) to a decrease in fluid and food intake and body weight gain, ii)  to the development of microcytic-hypochromic anaemia in the animals with excessive iron disposition in liver and spleen, unaltered plasma iron level and enhanced zinc concentration in red blood cells, characterized by a reduced haemoglobin level and haematocrit, unchanged counts of erythrocytes and reticulocytes, lowered total iron-binding capacity and elevated transferrin saturation, iii) disturbed Cu homeostasis, but iv) did not influence the leucocyte count and the plasma total antioxidant status.

 

Scibior et al. (2018): Vanadium alone did not significantly alter the thiobarbituric reactive substances and the activity of superoxide dismutase compared with the control but reduced slightly the glutathione reductase activity and the L-ascorbic acid level. It also markedly lowered the activity of catalase and glutathione peroxidase but elevated the activity of glutathione transferase and the hepatic L-ascorbic acid level to some degree.

Further, the vanadium concentration in the bone of the treatment group increased, whereas that of magnesium decreased compared with those in the control group. The total content of zinc and calcium did not change markedly in the treatment group compared to the control group.

In conclusion, the results provided evidence that the concentration of vanadium did not enhance the lipid peroxidation in rat bones. Vanadium administered disrupted the antioxidant defence mechanisms and homeostasis of some metals in bone tissue, which may consequently affect the balance in the activities of osteoblastic and osteoclastic cells.

 

Scibior & Zaporowska 2007: Outbred 2-month old male albino Wistar rats received for a period of 12 weeks vanadium metavanadate in drinking water at a concentration of 0.1 mg V/mL (equivalent to 8.63 mg V/kg bw/day). The control group received deionized drinking water. Food, fluid and water intakes were monitored daily and rats were weighed weekly. After 12 weeks, liver and kidney weights were recorded and the content of L-ascorbic acid in liver and kidney was analysed. Further, GSH and GSSG were determined in liver and kidney. The content of vanadium (V) in feed was analysed by ICP-AES. V, Fe, Zn and Cu were determined in liver and kidney by ICP-AES. Significant effects on GSH and GSSG level and GSH/GSSG ratio were observed in liver and kidney. Moreover, relative weights of liver and kidney were significantly increased. However, the slight effect on organ weights is considered to be small and thus an adaptive but not an adverse effect.  Thus, based on the adverse effects on GSH and GSSG level and GSH/GSSG ratio in liver and kidney, the LOAEL of this study is considered to be 8.63 mg V/kg bw/day. This study is well reported, however, no guideline was followed and only limited parameters were analysed. As no details on food and water intake were reported, it remains unclear if the palatability of vanadium containing drinking water was decreased. The author reported that GSH and GSSG level were slightly but significantly decreased in liver and kidney and vanadium levels in both organs were significantly increased. The same author reported a few years before that the levels of L-ascorbic acid were decreased in liver, spleen, kidney and adrenals as well as in plasma and erythrocytes (Zaporowska 1993, 1994). Both, L-ascorbic acid and GSH/GSSG (a disulfide derived from two glutathione molecules), are involved in detoxifying pentavalent vanadium compounds (as discussed in Zaporowska 1993, 1994). As shown in this study, vanadium was present in liver and kidney while in both organs levels of GSH and GSSG were reduced. Thus, the reported reduction of GSH and GSSG was most likley the result of an enhanced consumption/reduction activity of these compounds during detoxification of pentavalent vanadium. The fact that all cells are capable of synthesizing GSH and consumption of GSH initiates re-analysis of GSH most likely explains the slightly increased organ weights of liver and kidney. Thus, it is assumed that slightly affected organs weights were an adaptive but not an adverse effect.

 

Zaporowska et al. 1994: As reported in several other studies, effects on palatability of vanadium containing fluids were observed in high dose groups. Significant and dose-dependent decreases were observed in L-ascorbic acid levels in spleen, liver, adrenals and kidneys in males exposed to 0.15 and 0.30 mg V/mL and females exposed to 0.05, 0.15 and 0.3 mg V/mL. However, as the adversity of this effect remains questionable (discussed below) and no systemic toxicity was observed, this effect is not considered to be adverse and thus not taken into account for NOAEL setting. Based on this, 0.30 mg V/mL (equivalent to 22.06 mg V/kg bw/day in males and 26.62 mg V/kg bw/day in females) represents the NOEL. This study is well reported, however, no guideline was followed and only limited parameters were analysed. Significant and dose-dependent decreases were observed in L-ascorbic acid levels in spleen, liver, adrenals and kidneys in males exposed to 0.15 and 0.30 mg V/mL and females exposed to 0.05, 0.15 and 0.3 mg V/mL. Zaporwoska et al. (1993) reported already one year before that L-ascorbic acid levels were decreased in plasma and erythrocytes in rats exposed to ammonium metavanadate (see discussion above).

Scibior et al. 2005: Male Wistar rats were exposed to sodium metavanadate in drinking water (0.1 mg V/mL) for 6 weeks. Calculated average uptake of V was 8.35 ± 0.70 mg/kg/day. Food and fluid intake was reduced, which is however most likely a result of reduced palatability. Body weight gain was slightly affected (approx. 14 % reduction) but not significantly different compared to controls. An increase in erythrocyte count, a decrease in the mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin conc. (MCHC) was observed in the treatment group. No statistically significant decrease in the number of leukocytes, haematocrit and haemoglobin was observed in test animals. Based on these results, 8.35 mg V/kg bw/day is considered to be the lowest observed adverse effect level (LOAEL).

 

Scibior et al. 2006: In this study, vanadium at the dose of 10.7 mg/kg bw/d consumed by rats with their drinking water for 6 weeks caused a significant decrease in food and fluid intakes, body weight gain, RBC count, Hb level, and MCV and MCH values, whereas no significant differences in WBC count were observed in these animals compared to the control group. Additionally, vanadium at the tested dose resulted in a significant decrease in L-ascorbic acid concentration in plasma and caused a significant increase in MDA concentration in RBC, whereas no significant differences in Total Antioxidant Status level in plasma of rats were shown compared to the controls. Moreover, vanadium treated rats had a higher vanadium concentration in the plasma than the control animals. Based on these results, the lowest observed adverse effects level (LOAEL) is considered to be 10.69 mg V/kg bw/day.

 

Susic et al. 2006: The study was conducted in male rats with sodium metavanadate in the diet for 24 weeks. Groups of rats received 0, 300 or 3000 ppm NaVO3 in the diet. Investigations: systolic blood pressure, heart rate and body weight, renal functions, haematological parameters, cardiac output, total peripheral resistance, determination of plasma V concentration and weight of the right ventricular wall of the heart. Sub-chronic dietary treatment of rats with NaVO3 over 24 weeks (300 or 3000 ppm) did not affect blood pressure, but induced an increase in total peripheral resistance and a decrease in cardiac output in both groups. Haematocrit was significantly increased and plasma, blood and extracellular fluid volumes decreased in the high dose group. Renal function parameters were not affected by treatment of rats with sodium vanadate.

 

Higashino et al. 1983: Female SD rats either on normal or high potassium intake were exposed to 5 or 25 ppm vanadium metavanadate for 2 weeks. Body weight was recorded weekly and clinical signs as well as food intake were recorded. At study termination blood was collected and organ weights were recorded. Urine was examined twice for endogenous creatine and Na and K fractional excretion. Additionally, the vanadium concentration in different organs were analysed and the plasma concentration of Na and K was determined. Further, the specific activity of Na-K-ATPase in different tissues was determined. Vanadium exposure had no influence on clinical signs and food and water intake and body weight gain was not affected either. Haematological parameters and urinalysis were not affected by vanadium. Despite extremely high tissue levels of vanadium, no effect of the element on the basal activity of Na-K-ATPase could be observed. As no adverse effects, except elevated vanadium levels in different tissues, were observed, the NOAEL of this study is 25 ppm (equivalent to 2129.5 ± 91.2 meq/kg bw/day, highest concentration of rats exposed to normal potassium intake, high potassium concentration/intake was not considered here).

 

Conclusion

Altogether, available studies on oral tetra- and pentavalent vanadium compound exposure report significant and biological relevant effects on body weight, indicating that vanadium compounds induce systemic toxicity.

In addition, mild but significant effects on haematological parameter were consistently reported. Effects included reduced haemoglobin, reduced haematocrit, reduced mean cell haemoglobin concentrations, reduced mean corpuscular haemoglobin and mean corpuscular volume as well as elevated reticulocyte counts. Erythrocytes were consistently decreased except in the study of Scibior et al. 2005, which reported a slight but significant increase of erythrocytes in sodium metavanadate treated animals. In contrast, white blood cells were consistently unaffected. Although most of the reported effects on haematological parameters were only mild and in most cases within historical control ranges of the respective rat strain and age, the almost consistent reduction of erythrocytes and haematocrit and increase in reticulocytes indicates that tetra- and pentavalent vanadium compounds induce anaemic effects by reducing erythrocytes, which in turn triggers an erythropoietic response.

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

In addition to the haematological effects, Scibior and Zaporowska reported in several studies with sodium metavanadate or ammonium metavanadate, that the level of L-ascorbic acid and glutathione in plasma and erythrocytes as well as in different tissues is markedly decreased. The adversity of this effect remains unclear, as no other adverse effects were observed. However, a possible explanation for this significant reduction may be, that vanadium, known to be present in several tissues and body fluids, is detoxified by L-ascorbic acid and glutathione. Thus, the reduction is most likely the result of an enhanced consumption of these substances. However, as humans are not capable to synthesize L-ascorbic acid, it is unknown whether or not this effect is relevant for humans.

 

National Toxicology Programme

It is to be noted, that 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/). The NTP testing program foresees the conduct of animal studies via oral route (drinking water and feed) on VOSO4 & NaVO3 as follows:

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

-14 days dose-range finding experiments in Sprague-Dawley rats and B6C3F1/N mice (dose rats and mice: 0, 125, 250, 500, 1000, 2000 mg/L) – completed (Roberts et al. 2015)

- 90 days with Sprague-Dawley rats and B6C3F1/N mice (dose rats and mice: 0, 31.3, 62.5, 125, 250, or 500 ppm) – ongoing, first results presented below (Roberts et al. 2019)

- Perinatal dose-range finding study: gestation day 6 (GD 6) until postnatal day 42 (PND 42) with Harlan Sprague-Dawley rats - ongoing, first results presented below (Roberts et al. 2019)

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

It can reasonably be anticipated that these studies will be of high quality and relevance, and thus will serve as a more robust basis than the current data base with all its shortcomings.

 

In the study by Roberts et al. 2015, vanadium oxide sulfate was administered in drinking water at concentrations of 83.8, 167.5, 335, 670, and 1340 mg/L (equivalent to 11.7, 23.2, 35.6, 54.5, and 77.2 mg/kg/day for males and 10.0, 16.9, 28.4, 40.7, and 56.0 mg/kg/day for females). No mortality was observed at any dose, no clinical signs were observed in the 83.8, 167.5, and 335 mg/L groups, whereas clinical abnormalities (thin appearance, hunched posture, and ruffled coats) were seen in male and female mice in the 670 and 1340 mg/L groups, which were related to the body weight effects. At the 83.8, 167.5, and 335 mg/L groups group mean body weight of males and females were comparable with the vehicle group. Body weight reductions were observed in the 670 and 1340 mg/L groups, which however were significant only in the 1340 mg/L groups at study termination. Decreases in feed consumption were observed at 1340 mg/L in males and females, and were consistent with body weight effects. Water consumption per kilogram body weight (g/kg/day) decreased with increasing vanadium oxide sulfate concentration for both male and female mice. Although test item intake (mg/kg/day) increased with increasing concentration in drinking water, the increase was not dose proportional due to decreased palatability. Lastly, numerous statistically significant organ weight alterations were evident, although the vast majority of these alterations were proportional to, or at least followed the same trend as, terminal body weights. Reductions in absolute thymus and thymus-to-body weight ratios were apparent for male and female mice in the 1340 mg/L group, and in females in the 670 mg/L group, possibly due to stress associated with substance exposure and reduced water intake.

Overall, a confounding variable in this study is the reduced water consumption, which appears to be a cause of decreased palatability. The reduced water consumption observed in this study was accompanied with reduced body weight at the highest dose levels.

Due to the limited study design typical for a dose-range finding experiment (detailed clinical observations, haematology, clinical chemistry, and histopathology missing; limited organ weight determination) and severe confounder of reduced water intake, the study is used as supplementary information only.

Sodium metavanadate exposure in drinking water resulted in survival of 100 % of males and females in the 0, 125 and 250 mg/L groups, 80 % of males in the 500 mg/L group, and all females in the 500 mg/L group (125, 250 and 500 mg/L are equivalent to 18.364, 36.065, and 58.216 mg/kg/day for males and 15.247, 26.446, and 38.455 mg/kg/day for females, respectively). Due to early deaths, clinical observations, reduced body weights, and decreases in water and food consumption, animals in the 1000 and 2000 mg/L groups (equivalent to 59.587 and 109.377 mg/kg/day for males and 48.750 and 46.736 mg/kg/day for females, respectively) were terminated early on Day 8 (males) and Day 9 (females). Clinical abnormalities were limited to males and females in the 500, 1000 and 2000 mg/L groups, including shallow or rapid breathing, ruffled coat, abnormal gait, hunched posture, lethargy and/or thin appearance.

At study termination, body weight reduction was apparent in males and females in the 500 mg/L group. Reductions in food mean consumption (g/day) were no longer apparent when food consumption was normalized to body weight (g/kg/day) suggesting food intake was proportional to body weight at the 500 mg/L concentration. Effects on water consumption were apparent for males and females in the 500 mg/L groups, but when normalized to body weight, these effects indicated water consumption was proportional to body weight for these groups. Mice in the 1000 and 2000 mg/L groups exhibited more pronounced toxicity (i.e., mortality, moribundity, and clinical abnormalities) related to body weight effects whereas 80 % of males and 100 % of females in the 500 mg/L group survived to study termination. Thus, palatability issues may have contributed to body weight reductions in the 1000 and 2000 mg/L groups.

Organ weight alterations were noted in the heart, lungs, and ovary (right and left) across all exposure concentrations, whereas reductions in thymus and liver weights were noted in the 500 mg/L group only.

Overall, a confounding variable in this study is the reduced water consumption, which might be due to palatability, and the potential dehydration of the animals. Some of the clinical signs can be attributed to dehydration. In addition, lower water consumption might have caused the body weight reduction observed at the 2000 mg/L dose level.

Due to the limited study design, short exposure duration of only 14 days, restricted examination of the animals (detailed clinical observations, haematology, clinical chemistry, and histopathology missing; limited organ weight determination), and lack of reporting the individual data, the study will not be used for hazard and risk assessment purposes but as supplementary information.

In the study by Roberts et al. (2019), time-mated F0 Hsd:Sprague-Dawley rats were exposed to sodium metavanadate and vanadyl sulfate at concentrations of 0, 31, 63, 125, 250, and 500 mg/L and 0, 21, 42, 84, 168, and 335 mg/L, respectively via drinking water on GD 6. Groups of male and female F1 animals were exposed during gestation, lactation and 13-weeks post-weaning (5 animals for biological sampling). Dams/pups exposed to sodium metavanadate at 250 and 500 mg/L in drinking water exhibited moribundity at birth, failure to thrive, and higher moribundity during lactation. Lower body weights were observed in dams during gestation and lactation, and in pups continuing until study termination 13 weeks post-weaning. Vanadyl sulfate, up to 335 mg/L in drinking water, was well tolerated in time-mated rats during gestation and lactation, and their pups during lactation and up to 13-weeks post weaning.

In study by Roberts et al. (2019), groups of 10 male and 10 female B6C3F1/N mice were exposed to sodium metavanadate and vanadyl sulfate at concentrations of 0, 31, 63, 125, 250, and 500 mg/L and 0, 21, 42, 84, 168, and 335 mg/L, respectively via drinking water for a duration of 13 weeks. Sodium metavanadate in drinking water resulted in lower body weights and water consumption at 500 mg/L in adult mice. Mice exposed to higher concentrations of sodium metavanadate also had lower thymus weight. Lastly, mice exposed to higher (125, 150, 500 mg/L) concentrations of sodium metavanadate exhibited increase in erythrocytes and reticulocytes and decreases haematocrit and haemoglobin. Vanadyl sulfate, up to 335 mg/L in drinking water, was well tolerated in adult mice.

The results of NTP studies by Roberts et al presented above are not yet fully accessible or published, since the review process is still ongoing. The results presented above are based on a 2019 congress abstract/poster. A complete robust study summary will be provided in the dossier upon availability of the data.

 

 

Inhalation:

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

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

 

Human data: Regarding the preferential use of human data in risk assessments for human health, a respective statement is attached below. There are several epidemiological studies linking upper respiratory symptoms to vanadium pentoxide exposure (Kiviluoto, 1980; Kiviluoto et al., 1979a; Lewis, 1959, Zenz and Berg, 1967 Zenz et al. 1962). Long-term chronic exposure data of workers in the vanadium industry are reported in several publications. In a factory manufacturing vanadium pentaoxide, 63 workers exposed to V₂O₅at concentrations of 0.1 to 3.9 mg V/m³measured as total dust for 11 years (average 0.2-0.5 mg V/m³) did not have an increased prevalence of upper respiratory symptoms in the case study by Kiviluoto et al (1979a,b, 1980, 1981a,b).

Kiviluoto et al. (1979b) did not observe any differences in the anterior and posterior rhinoscopy in the exposed groups after 11 years of exposure to average V₂O₅levels of 0.2-0.5 mg V/m³as listed above. Furthermore, there was no difference in the number of blood vessels between the exposed and non-exposed groups. However, the number of neutrophils in the nasal smears and the number of plasma cells in the nasal mucosa were increased indicative of a protective mechanism in the mucosa. Other examined factors of the biopsies and cell findings did not differ between the exposed workers and the controls. Chest radiographs and lung function tests did not reveal any differences. After further 7-11 months of V₂O₅exposure at concentrations ranging from 0.01 to 0.04 mg V/m³measured as total dust, a subsequent reexamination revealed that the cell findings did not indicate any further significant changes between the studied exposed groups, and that there were no significant changes in the number of eosinophils of cytological and histological samples.

Altogether, no pneumoconiosis and no other signs indicative of allergic inflammation, including nasal catarrh, cough, phlegm, were observed by Kiviluoto et al. in the exposed subjects working for 11 years under these occupational conditions.

 Other epidemiological data support that respiratory symptoms are observed at exposure concentrations of V₂O₅that are above 0.1 mg/ V/m³, and are summarized in the following table

 

Table: Epidemiological studies ofV₂O₅exposure

Subjects	V2O5Dose [mg V /m^3]	Exposure duration	Symptoms	study
24 workers	0.1 - 0.93 mean PS < 5 μm		eye, nose, throat irritation; cough; wheezing, nasal mucosa, rales, rhonchi; injected pharynx and green tongue	Lewis, 1959
2 volunteers	1	8 h	cough, no eosinophilia, normal white blood cell count & cell patterns, no effects on urinalysis, normal lung function	Zenz & Berg, 1967
5 volunteers	0.2 (PS: 98 % < 5 μm)	8 h	loose cough, no eosinophilia, normal white blood cell count & cell patterns, no effects on urinalysis, normal lung function, no detectable V in the blood	Zenz & Berg, 1967
2 volunteers	0.1	8 h	formation of mucus	Zenz & Berg, 1967
3 of 18 workers	> 0.5 mean PS < 5 μm	24 h	inflamed throat, dry cough, burning eyes, no wheeze	Zenz et al. (1962)
11 volunteers	0.4 condensation aerosol		tickling, itching, dryness of mouth mucosa	Pazhynic, 1967
5 of 11 volunteers	0.16		mild signs of irritation	Pazhynic, 1967
11 volunteers	0.08		no notice of symptoms	Pazhynic, 1967
8 volunteers (4 workers + 4 trainees)	0.028 – 0.062	8 h/d, 5 d	no notice of symptoms (i.e. neurobehavioural, neuro-psychological, psychosomatic & psychological effects)	Hörtnagl et al. 1994

 

The Scientific Committee on Occupational Exposure Limit summarized these studies as follows:„In workers exposed to dust containing vanadium (as vanadium pentaoxide) 0.2-0.5 mg/m³for about 11 years, irritants effects on the mucous membranes of the upper respiratory tract were reported. After hygienic improvements, the same workers were exposed to VP concentrations in the range of 0.01-0.04 mg/m³for about 10 months. No worsening of the irritant effects observed as a consequence of the previous exposure was reported for this low-level exposure. In these workers, the exposure did not cause any pathological effects on the blood picture, the cysteine level in the hair, or the respiratory function (Kiviluoto et al., 1979a,b, 1980, 1981a,b; Kiviluoto, 1980)…

Kiviluoto et al (1979a,b, 1980, 1981a,b): in their studies on 63 males exposed in a vanadium factory for 11 years at concentrations in the range of 0.1-3.9 mg/m³(estimated average concentrations 0.2-0.5 mg/m³) and after a further 7-11 month later when concentrations had been reduced to 0.01-0.04 mg/m³studied nasal smears and biopsies. The findings were consistent with irritant effects. Eosinophils did not differ between exposed and non-exposed, nor did IgE-antibody levels. Although exposed workers complained significantly more often of wheezing, pulmonary function tests did not differ. There is, thus, little evidence indicating sensitizing effects on the respiratory tract. The known irritant effects of VP can well explain effects on the respiratory tract including rhinitis, bronchial hyper-reactivity, wheeze, asthma as well as bronchitis…

 

For respiratory tract irritation, and more generally speaking for upper and lower airways effects, dose-response relationships could be obtained in both experimental animals and humans. It can be assumed that 0.04 mg/m3 has to be considered as a NOEL in occupationally exposed subjects (10 months), while in rodents a NOEL could be concluded at an exposure level of 2 mg/m³(B6C3F1 mice, m. f., inhalation, 6h/day, 5d/w for 16 days) and of 1 mg/m3 (F344/N rats, m. f., inhalation, 6h/day, 5d/w for 14 weeks)…

It appears that exposure to concentrations <0.1 mg/m³do not induce irritating effects on the respiratory tract.(SCOEL/SUM/62 Final, January 2004)“

Evidence from animal and human data suggests that exposure to elevated V₂O₅concentrations may result in irritating effects on the respiratory system. However, human data were used as point of departure for the DNEL derivation because long-term chronic data are available from workers exposed to vanadium dust using a sensitive indicator of irritation (cytology), a population similar to the target population (workers of the vanadium industry), and to decrease uncertainty for interspecies differences in sensitivity.

Therefore, the NOAEC of 0.04 mg V /m³(measured as total dust) for humans exposed occupationally for 11 years to V₂O₅dust was used as POD for the DNEL derivation.

 

Read-across:There is a complete lack of studies that would allow distinguishing whether or not local effects of V₂O₅are relevant for other vanadium substances. Based on the assumption that irritancy of a particular V substance is the driver for local effects, read-across of irritating effects of V₂O₅to other soluble vanadium substances was assessed. Five substance-specific properties are assumed to predominantly account for the observed irritation potential of V₂O₅: (i) water solubility and potential to become bioavailable, (ii) acidifying properties in aqueous media and potential for related burning, (iii) oxidising properties and potential for oxidative injury, (iv) irritating / corrosive properties (skin and/or eye) and potential for irritation of mucous membranes, and (v) particle size distribution and potential for inhalability. Vanadium, oxalate complexes is assessed as follows: 

(i) Vanadium, oxalate complexes with a water solubility of 145.1 ± 11.4 g/L at pH 1.9 / 20°C is considered very soluble.

(ii) Vanadium, oxalate complexes has an acidifying effect as in the saturated aqueous solution (i.e. water solubility test), a pH of 1.9 was measured.

(iii) Vanadium, oxalate complexes is void of oxidising properties and the potential for oxidative injury.

(iv) Where available data indicates any irritating effects either to skin, eye or respiratory tract, it was assumed in a conservative approach that the respective substance may have an irritation potential. Hence, vanadium, oxalate complexes is considered as a substance with irritation potential.

(v) The inhalability as an additional modifying factor was derived from particle size distributions (i.e., granulometry), for which all vanadium substances were subjected to an experimental testing programme. Physical particle size distributions of commercial materials were determined experimentally and are represented by the median particle size diameter (d50).

Since “physical” particle size distributions do not necessarily reflect the particle size of aerosols that may be formed under practically relevant workplace conditions (e.g., during manual operations, including bag filling and emptying, or under mechanical agitation during mixing and weighing), the particle size distribution of the airborne fraction generated during mechanical agitation in the rotating drum according to the method by Heubach (1991) was additionally determined according to DIN 55992 Part 1. Furthermore, using the mass fractions deposited on the impactor stages, mass median aerodynamic diameter (MMAD) of the airborne material and geometric standard deviation (GSD) of the MMAD were determined (Grewe, 2010 & 2013). The Multiple Path Particle Deposition (MPPD) model (CIIT, 2002-2009) was applied to estimate the deposition of particles in the respiratory tract (head, tracheobronchial and pulmonary region) of workers. As a result, divanadium tris(sulphate) was assessed as being (at least partly) inhalable based on MPPD model outputs using worker specific input parameters as well as MMAD and GSD estimates.

 

If a V substance possesses four or more of the five substance-specific properties assumed to predominantly account for the observed irritation of V₂O₅, read-across of irritating effects of V₂O₅is considered to be justified in a conservative approach (see Table below).

 

	potential to become bioavailable	potential for acidic burning	potential for inhalability	potential for oxidative injury	potential for irritation of mucous membranes	Read-across of irritation
VO(C2O4)	yes	yes	*	no	yes	yes

* VO(C2O4) is manufactured and sold in solution only and as such are not inhaled. However, for pre-cautionary considerations, the solid product is being assessed and inhalability was nevertheless assumed.

 

As already stated, the applied approach is likely to overestimate the potential of vanadium, oxalate complexes to irritate mucous membranes. However, in the absence of further data, a differentiation cannot be scientifically supported and vanadium, oxalate complexes is conservatively assumed to have a potential to irritate the respiratory tract following repeated exposure. Nevertheless, no carcinogenicity, no pneumoconiosis and no other signs indicative of allergic inflammation have been reported for workers manufacturing vanadium, oxalate complexes.

 

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

(i) chronic toxicity,

(ii) carcinogenicity, and 

(iii) multi-generation reproductive toxicity

is planned.

 

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

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

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

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

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

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

 

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

 

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

A number of studies are available where vanadium compounds were administered; however, they have involved different experimental approaches and designs as well as different dose regimens, and endpoints. For oral exposure, effects are more limited and the different experimental approaches lead to a variety of endpoints measured.

 

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:

reliable GLP-conform study with V2O5

 

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:

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

 

References:

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

 

Justification for selection of repeated dose toxicity dermal - local effects endpoint:

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

 

References:

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

Justification for classification or non-classification

Based on read-across of V2O5 data and in a conservative approach, local effects are considered relevant and vanadium, oxalate complexes is assumed to irritate the respiratory tract following repeated exposure. Therefore, vanadium, oxalate complexes (in the solid but not in the soluble form) meet the classification criteria for Specific target organ toxicity-repeated exposure - Category 1 according to Regulation (EC) No 1272/2008.