Divanadium trioxide

Endpoint summary

Description of key information

Inhalation:

A study via the inhalation route is available for divanadium trioxide. However, data via the oral route are not available for divanadium trioxide but exist for other vanadium substances. The rationale for read-across to divanadium trioxide including the limitations thereof is summarised below (see discussion).

In a 14-d repeated-dose inhalation toxicity study, Sprague-Dawley rats were exposed to micronised vanadium trioxide powder via nose-only inhalation. Test-substance related mortality or any clinical signs of systemic toxicity were not observed at any exposure level. Body weights/body weight gain and food consumption were significantly decreased only at the highest exposure (0.25 mg/L) in males and females. In summary, based on the local effects in the respiratory tract of the test animals at the high exposure level (0.25 mg/L), the NOAEC was 0.02 mg/L (20 mg V2O3/m3). All effects on BAL parameters, lung weights and lung histopathology seen at the lower exposure levels were considered as adaptive responses to divanadium trioxide exposure, rather than an indication of local toxicity.

Information on repeated dose toxicity following inhalation exposure to V2O5 is available in a NTP study (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. However, local effects on the respiratory tract are not considered relevant for divanadium trioxide.

Oral:

In an unpublished study report with vanadium carbide nitride, relevant treatment-related effects were not observed in rats after 28-days oral exposure up to the limit dose of 1000 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-04-07 to 2011-07-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 1995-07-27

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2010-01-26

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 44 to 50 days
- Weight at study initiation: males: 238 to 294 g; females: 165 to 242 g
- Housing: the animals were housed five of one sex per cage. The cages were made of a polycarbonate body with a stainless steel mesh lid. Wood based material was used as bedding and was sterilised by autoclaving
- Diet (ad libitum, except when urine was being collected and overnight before routine blood sampling): a standard rodent diet (Rat and Mouse No. 1 Maintenance Diet)
- Water (ad libitum, except when urine was being collected): potable water
- Acclimation period: 15 days

Each cage of animals was provided with an Aspen chew block for environmental enrichment. Chew blocks were provided throughout the study. Each
cage of animals was provided with a plastic shelter for environmental enrichment.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 23°C
- Relative humidity: 40 to 70%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was prepared for administration as a series of suspensions in the vehicle. For each test formulation the required amount of test material was weighed and transferred to a suitably sized mortar to be ground into a fine powder. Small amounts of the pre-weighed vehicle were added and mixed with the test material using a pestle, ensuring any agglomerates were broken down to produce a smooth paste. The suspension was poured into a measuring cylinder which had been wetted with vehicle. The mortar was thoroughly rinsed with vehicle and this was added to the cylinder. The required volume was achieved with the remaining vehicle. The suspension was transferred into a beaker and mixed using a high shear homogeniser until it was homogenous. Finally, the suspension was transferred into containers, via syringe, whilst magnetically stirring.
The test substance was used as supplied. All formulations were prepared weekly and stored refrigerated (approximately 2-8°C) until use.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
30 mg/kg/day: concentration: 6 mg/mL; volume dose: 5 mL/kg
300 mg/kg/day: concentration: 60 mg/mL; volume dose: 5 mL/kg
1000 mg/kg/day: concentration: 200 mg/mL; volume dose: 5 mL/kg
The volume administered to each animal was calculated from the most recently recorded bodyweight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before treatment commenced, the suitability of the proposed mixing procedure was determined and specimen formulations were analysed to assess the homogeneity and stability of the test substance in the liquid matrix.
Samples of each formulation prepared for administration in Week 1 of treatment were analysed for achieved concentration and homogeneity of the test substance.

Methods:
1) Homogeneity and stability in corn oil formulations
The homogeneity and stability of vanadium carbide nitride in corn oil formulations was assessed at nominal concentrations of 2 mg/mL and 200 mg/mL, during ambient and refrigerated storage. Freshly prepared specimen formulations (400 mL) were equally sub-divided (4 × 100 mL) into four amber glass screw top bottles and submitted for analysis.
- Ambient temperature storage (nominally +21ºC): on receipt, the contents of one bottle of each formulation were mixed by 20-fold inversion followed by vigorous shaking (30 seconds) and magnetic stirring. After stirring for 5 minutes (representing 0 hour), 1 hour and 2 hours, single samples (nominally 1 mL) were removed for analysis from approximately one quarter, one half and three quarters the depth (representing the top, middle and bottom) of the continuously stirred formulation.
The remainder of the bottle was stored at ambient temperature and after 1 day storage the contents were remixed and sampled as detailed above.
- Refrigerated storage (nominally +4ºC): the remaining bottles were refrigerated on receipt and on Days 1, 8 and 14, the appropriate bottle was removed from storage and equilibrated to ambient temperature. The contents of the bottle were mixed by 20-fold inversion followed by vigorous shaking (30 seconds) and
magnetic stirring for 5 minutes and single samples (nominally 1 mL) were removed for analysis from the top, middle and bottom of the stirred formulation.

2) Concentration in test formulations
At Week 1 of treatment, freshly prepared test formulations were sampled (1 mL, accurately weighed from the top, middle and bottom strata) and submitted for
analysis. The concentrations of vanadium carbide nitride was analysed by atomic absorption spectroscopy technique using the graphite furnace.

Results:
The homogeneity and stability was confirmed for vanadium carbide nitride in corn oil formulations at nominal concentrations of 2 mg/mL and 200 mg/mL for ambient temperature storage for 1 day and refrigerated storage for up to 8 days. The storage times represented the maximum time from preparation to completion of administration.
The mean concentrations of vanadium carbide nitride in test formulations analysed for Week 1 of the study were within +10%/-15% of nominal concentrations, confirming accurate formulation.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

once daily, seven days per week

Remarks:

Doses / Concentrations:
0, 30, 300 and 1000 mg/kg /day
Basis:
actual ingested

No. of animals per sex per dose:

Main study and recovery groups:
0 mg/kg/day: 5 males / 5 females (plus 5 males / 5 females as recovery group)
30 mg/kg/day: 5 males / 5 females
300 mg/kg/day: 5 males / 5 females
1000 mg/kg/day: 5 males / 5 females (plus 5 males / 5 females as recovery group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The results of a preliminary study conducted be the laboratory (Study number IVY0008) at doses of 250, 500 or 1000 mg/kg/day showed that the test substance was well tolerated, with no findings considered to be related to treatment at any dose. It was therefore considered that a high dose of 1000 mg/kg/day would be appropriate for the present study, with low and intermediate doses at 30 and 300 mg/kg/day, respectively.

- Post-exposure recovery period in satellite groups: animals assigned to the recovery phase completed a further two weeks without treatment.

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of ill-health amongst the occupants.
Daily during the study detailed observations were recorded at the following times in relation to dose administration:
Week 1 of treatment: immediately before dosing, on completion of dosing of each group, between one and two hours after completion of dosing of all groups, and as late as possible in the working day
Weeks 2 to 4 of treatment: immediately before dosing and between one and two hours after completion of dosing of all groups
Week 1 and 2 of recovery: at approximately the same time each day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before treatment commenced and during each week of treatment and recovery, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period).
After removal from the home cage, animals were assessed for physical condition and behaviour during handling and after being placed in a standard arena. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behaviour.

BODY WEIGHT: Yes
- Time schedule for examinations: the weight of each rat was recorded before treatment commenced (Day -7), on the day that treatment commenced (Day 1), on Days 8, 15, 22 and 28 of the treatment period and on Days 1, 8 and 14 of the recovery period and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for each week throughout the treatment and recovery periods. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: fluid intake was assessed by daily visual observation. No effect was observed and, consequently, quantitative measurements were not performed.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on Day 29 of treatment (after the 28th dose), blood samples were obtained from all main study animals. On Day 15 of recovery blood samples were obtained from all recovery phase animals. The blood samples were withdrawn from the sublingual vein.
- Anaesthetic used for blood collection: Yes, light general anaesthesia induced by isoflurane
- Animals fasted: Yes, the samples were taken after overnight withdrawal of food.
- How many animals: all main study and all recovery phase animals
- Parameters examined: haematocrit, haemoglobin concentration, erythrocyte count, reticulocyte count, mean cell haemoglobin, mean cell haemoglobin concentration, mean cell volume, total white cell count, differential WBC count (neutrophils, lymphocytes, eosinophils, basophils, monocytes and Large unstained cells), platelet count, prothrombin time and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on Day 29 of treatment (after the 28th dose), blood samples were obtained from all main study animals. On Day 15 of recovery blood samples were obtained from all recovery phase animals. The blood samples were withdrawn from the sublingual vein.
- Animals fasted: Yes, the samples were taken after overnight withdrawal of food.
- How many animals: all main study and all recovery phase animals
- Parameters examined: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, urea, creatinine, glucose, total cholesterol, total bile acids, triglycerides, sodium, potassium, chloride, calcium, inorganic phosphorus, total protein and albumin
Albumin/globulin ratio was calculated from total protein concentration and analysed albumin concentration.

URINALYSIS: Yes
- Time schedule for collection of urine: during Week 4 of treatment overnight urine samples were collected from all main study animals and during Week 2 of recovery overnight urine samples were collected from all recovery phase animals.
- Metabolism cages used for collection of urine: Yes, animals were placed in an individual metabolism cage.
the following day.
- Animals fasted: Yes
- Parameters examined: appearance, volume, pH, specific gravity, protein, glucose, ketones, bile pigments, and blood pigments
A microscopic examination of the urine sediment was performed (epithelial cells, leucocytes, erythrocytes, crystals, spermatozoa, casts and abnormalities).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: sensory reactivity and grip strength assessments were performed (before dosing), during Week 4 of treatment.
During Week 4 of treatment (before dosing), the motor activity was measured and In addition, all recovery phase animals were tested during Week 2 of recovery.
- Dose groups that were examined: 30 and 300 mg/kg/day (main study) and all recovers phase animals
- Battery of functions tested:
The following measurements, reflexes and responses were recorded:
1) Sensory reactivity: approach response, touch response, auditory startle reflex, tail pinch response,
2) Grip strength
3) Motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Animals surviving until the end of the scheduled treatment or recovery period were killed by carbon dioxide asphyxiation followed by subsequent exsanguination. The sequence in which the animals were killed after completion of treatment or recovery was selected to allow satisfactory inter-group comparison.
All animals were subject to a detailed necropsy.
After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. The cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded.
The requisite organs were weighed and external and cut surfaces of the organs and tissues were examined as appropriate. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.

ORGAN WEIGHTS:
The following organs were taken from each animal killed after four weeks of treatment or two weeks of recovery and weighed: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles, spleen, testes, thymus and uterus with cervix
Prostate and seminal vesicles with coagulating gland were weighed together.
Bilateral organs were weighed together. Organ weights were also adjusted for terminal bodyweight, using the weight recorded before necropsy.

HISTOPATHOLOGY: Yes
Microscopical examination of the following organs: adrenals, ovaries, brain, Peyer’s patches, caecum, pituitary, colon, prostate, duodenum, rectum, epididymides, eyes, seminal vesicles, heart, ileum, spinal cord, jejunum, spleen, kidneys, sternum, stomach, liver, testes, lungs, thymus, lymph nodes (mandibular, mesenteric, left axillary), thyroid with parathyroids, trachea, urinary bladder, uterus and cervix, and vagina
Microscopic examination was performed as follows:
- all tissues preserved for examination were examined for all animals of vehicle control group and the 1000 mg/kg/day dose level group sacrificed on completion of the scheduled treatment period.
- tissues reported at macroscopic examination as being grossly abnormal were examined for main and recovery animals in line with current practice.
Those tissues subject to histological processing included the following regions:
- adrenals: cortex and medulla
- brain: cerebellum, cerebrum and midbrain
- femur with joint: longitudinal section including articular surface, epiphysial plate and bone marrow
- heart: included auricular and ventricular regions
- kidneys: included cortex, medulla and papilla regions
- liver: section from two main lobes
- lungs: section from two major lobes, to include bronchi
- spinal cord: transverse and longitudinal section at the cervical, lumbar and thoracic levels
- stomach: included keratinised, glandular and antrum in sections
- thyroid: included parathyroids in section where possible
- uterus: uterine body with cervix section
For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required for microscopic pathology

Statistics:

All statistical analyses were carried out separately for males and females. All analyses were carried out using the individual animal as the basic experimental unit.
The following data types were analysed at each time point separately: grip strength and motor activity, bodyweight (using gains over appropriate study periods), blood chemistry, haematology and urinalysis, organ weights.
The following statistical tests were used for grip strength, motor activity, bodyweight, organ weight and clinical pathology data: Bartlett's test for variance homogeneity, t-tests, F1 approximate test, Williams’ test, Dunnett's test, Wilcoxon’s rank sum tests, H1 approximate test, Shirley's test, Steel's test, Fisher’s Exact tests and analysis of covariance
Significant differences between Control and treated groups were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no deaths.
The appearance and behaviour of the animals were unaffected by treatment.

BODY WEIGHT AND WEIGHT GAIN
Overall bodyweight gain was unaffected by treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Overall food consumption was unaffected by treatment.

WATER CONSUMPTION AND COMPOUND INTAKE
Visual assessment of water intake did not identify an effect of treatment.

HAEMATOLOGY
The haematological investigation on Day 29 revealed, when compared to the controls, slightly low total white cell counts (associated with slightly low lymphocyte counts) for females at 300 or 1000 mg/kg/day. Basophil and monocyte counts were also slightly low for females at 1000 mg/kg/day. These differences from controls were still apparent on Day 15 of the recovery period for females previously treated at 1000 mg/kg/day.
Activated partial thromboplastin times were also slightly reduced, when compared to the controls, for females at 300 or 1000 mg/kg/day. This difference from controls was not apparent on Day 15 of the recovery period for females previously treated at 1000 mg/kg/day.
Minor changes seen in blood parameters were considered to be of minor toxicological importance and not adverse in nature.

CLINICAL CHEMISTRY
The biochemical examination of the blood plasma on Day 29 revealed, when compared to the controls, slightly low bile acid levels for males at 300 or 1000 mg/kg/day. Phosphorus levels were also slightly low for males at 1000 mg/kg/day. These differences from controls were no longer apparent on Day 15 of the recovery period for males previously treated at 1000 mg/kg/day.
Minor changes seen in blood parameters were considered to be of minor toxicological importance and not adverse in nature.

URINALYSIS
The appearance and composition of urine was not affected by treatment.

NEUROBEHAVIOUR
There were no effects on sensory reactivity or grip strength that were considered to be related to treatment.
There were no effects on motor activity that were considered to be clearly associated with treatment.

ORGAN WEIGHTS
The assessment of organ weights after 4 weeks of treatment revealed, when compared to the controls, slightly high adrenal weight and slightly low spleen weight for females at 1000 mg/kg/day. These inter-group differences were not apparent in females previously treated at 1000 mg/kg/day killed after 2 weeks of recovery.
Minor changes seen in organ weights were considered to be of minor toxicological importance and not adverse in nature.

GROSS PATHOLOGY
There were no treatment-related macroscopic findings seen at necropsy.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no treatment-related histopathology changes seen at 1000 mg/kg/day.

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

not specified

Conclusions:

In the study by Chase (2011), groups of 5 male and 5 female Sprague Dawley rats were given vanadium carbide nitride at doses of 0, 30, 300, and 1000 mg/kg bw/day via gavage. Recovery animals (5M/5F) were added for the control and high dose group with a scheduled sacrifice 14-days post exposure. The oral administration of vanadium carbide nitride was generally well tolerated, showing no effects on body weight, body weight gain and food/water consumption. No clinical signs of intolerance and no effects on neurobehaviour (sensory activity, grip strength, motor activity) were observed.
Slightly low white blood cell counts were noted in females at 300 and 1000 mg/kg/day. The differences from controls were small, males were not similarly affected and there were no pertinent histopathological changes seen in the lymphoid tissue or bone marrow. Similarly, at 300 and 1000 mg/kg/day, the slightly reduced activated partial thromboplastin times for females and low bile acid levels for males were small, seen in one sex only and were not associated with any histopathological changes in the liver. The inter-group differences at 1000 mg/kg/day were also not apparent after 2 weeks of recovery. The values were still within the reference interval for (female) Sprague Dawley rats reported by He et al. (2017) and Lillie et al. (1996). In the absence of corroborative histopathology, the above findings were considered to be of minor toxicological importance and not adverse in nature.
The oral administration of Vanadium Carbide Nitride to Crl:CD(SD) rats for 4 weeks at doses up to 1000 mg/kg/day was generally well tolerated. There were no relevant adverse effects observed in any of the parameters investigated. Consequently, the No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-10-19 to 2011-11-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

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

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

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

GLP compliance:

no

Remarks:

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

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

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

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

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Remarks on MMAD:

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

Details on inhalation exposure:

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

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

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

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

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

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

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Please refer to "Details on inhalation exposure" above.

Duration of treatment / exposure:

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

Frequency of treatment:

6 hours per day, 5 days per week

Dose / conc.:

0.002 mg/L air (analytical)

Dose / conc.:

0.022 mg/L air (analytical)

Dose / conc.:

0.25 mg/L air (analytical)

No. of animals per sex per dose:

6 males / 6 females

Control animals:

yes, concurrent vehicle

Positive control:

none

Observations and examinations performed and frequency:

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

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

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

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

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

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

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

Sacrifice and pathology:

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

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

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

Statistics:

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

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

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

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

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

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

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

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

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

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

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

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

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

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

Dose descriptor:

NOAEC

Effect level:

0.022 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Dose descriptor:

LOAEC

Effect level:

0.25 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Critical effects observed:

not specified

Conclusions:

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-10-19 to 2011-11-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

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

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

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

GLP compliance:

no

Remarks:

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

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

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

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

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Remarks on MMAD:

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

Details on inhalation exposure:

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

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

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

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

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

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

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Please refer to "Details on inhalation exposure" above.

Duration of treatment / exposure:

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

Frequency of treatment:

6 hours per day, 5 days per week

Dose / conc.:

0.002 mg/L air (analytical)

Dose / conc.:

0.022 mg/L air (analytical)

Dose / conc.:

0.25 mg/L air (analytical)

No. of animals per sex per dose:

6 males / 6 females

Control animals:

yes, concurrent vehicle

Positive control:

none

Observations and examinations performed and frequency:

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

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

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

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

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

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

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

Sacrifice and pathology:

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

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

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

Statistics:

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

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

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

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

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

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

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

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

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

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

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

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

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

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

Dose descriptor:

NOAEC

Effect level:

0.022 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Dose descriptor:

LOAEC

Effect level:

0.25 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Critical effects observed:

not specified

Conclusions:

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

20 mg/m³

Study duration:

subacute

Species:

rat

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

Read-across - inhalation:

The bioavailability and reactivity of V2O3 in tissues of the respiratory tract is assumed to be far less than that of V2O5. Thus, read-across from very soluble pentavalent vanadium substances to poorly/less soluble and poorly/less bioavailable for the purpose of classification and labelling of V2O3 is not supported.

 

Read across – oral:

The endpoint repeated dose toxicity of the vanadium category substance is not addressed by substance-specific information but rather by read-across of data available for soluble tetra- and pentavalent vanadium substances as well as for insoluble vanadium substances with zero valency. 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. For further details on the read-across approach for the oral/systemic effects of the vanadium category substances and details on the read-across groups of the soluble and poorly soluble vanadium substances, please refer to the report attached to section 13 of the technical dossier. Therefore, the remaining text in this chapter is generic for all vanadium substances and has not been adapted on a substance-specific basis.

Oral:

In the study by Chase (2011), groups of 5 male and 5 female Sprague Dawley rats were given vanadium carbide nitride at doses of 0, 30, 300, and 1000 mg/kg bw/day via gavage. Recovery animals (5M/5F) were added for the control and high dose group with a scheduled sacrifice 14-days post exposure. The oral administration of vanadium carbide nitride was generally well tolerated, showing no effects on body weight, body weight gain and food/water consumption. No clinical signs of intolerance and no effects on neurobehaviour (sensory activity, grip strength, motor activity) were observed.

Slightly low white blood cell counts were noted in females at 300 and 1000 mg/kg/day. The differences from controls were small, males were not similarly affected and there were no pertinent histopathological changes seen in the lymphoid tissue or bone marrow. Similarly, at 300 and 1000 mg/kg/day, the slightly reduced activated partial thromboplastin times for females and low bile acid levels for males were small, seen in one sex only and were not associated with any histopathological changes in the liver. The inter-group differences at 1000 mg/kg/day were also not apparent after 2 weeks of recovery. The values were still within the reference interval for (female) Sprague Dawley rats reported by He et al. (2017) and Lillie et al. (1996). In the absence of corroborative histopathology, the above findings were considered to be of minor toxicological importance and not adverse in nature.

The oral administration of Vanadium Carbide Nitride to Crl:CD(SD) rats for 4 weeks at doses up to 1000 mg/kg/day was generally well tolerated. There were no relevant adverse effects observed in any of the parameters investigated. Consequently, the No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day or above.

 

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, effects noted have included reduced haemoglobin, reduced haematocrit, reduced mean cell haemoglobin concentrations, while effects on red blood cells have included both reductions and increases depending on dose levels used and duration of treatment, perhaps compensating for the haemoglobin effect. 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.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 the appropriate critical effect for oral exposure from the available dataset. Additional support for the reliability of this endpoint comes from a study by Hogan (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)).

 

 

Inhalation:

The most informative study is the standard NTP chronic inhalation carcinogenicity 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.

 

However, local effects on the respiratory tract are not considered relevant for divanadium trioxide for the following reasons:

Severe irritant properties of V2O5 have been identified for eye (cat 1) and in lungs, and the redox potential of V2O5 as well as the sharp decline on pH in contact with aqueous media is hypothesised to either mediate this mechanism or at least propagate this mechanism. In contrast, there is no indication whatsoever of any potential for irritation of the respiratory tract for V2O3. With regard to substance-specific properties assumed to predominantly account for an irritation potential, V2O3 is different from V2O5 as follows:

- A low dissolution of V2O3 was observed in artificial lysosomal fluid (11.9% after 2h; 15.7% after 24h) and lung fluid (4.7% after 2h; 5.6% after 24h)while pentavalent substances (V2O5or NaVO3) dissolved completely within 2 h. Thus, the bioavailability and reactivity of V2O3 in tissues of the respiratory tract is assumed to be far less than that of V2O5.

- V2O3 upon contact with water does not cause such significant pH decrease as is the case for V2O5, thus indicating a lack of acidifying properties in aqueous media and any potential for tissue injury associated therewith of V2O3.

- V2O3 is completely void of oxidising properties and the potential for oxidative injury.

- Only some very mild but reversible effects have been observed in vivo in the eye after exposure to V2O3.

-V2O3 is not acutely toxic or harmful via inhalation whereas V2O5 is.

Regarding the potential for respiratory irritation, a comprehensive histopathological evaluation of lung tissue was performed within 14-d inhalation studies conducted both with V2O3 and V2O5. Severe lung effects including hyperplasia in alveolar and bronchial epithelia, inflammation or fibrosis could not be observed at exposure levels up to 250 mg/m3 with V2O3, whereas these effects are reported as severe for all animals exposed to V2O5 already at a level of 2 mg/m3. In conclusion, the onset of marked irritation effects with V2O5 occurs at exposure levels approx. 100-fold lower than with V2O3; on the other hand, given the low solubility and the high exposures, the onset of overload phenomena cannot be completely excluded for V2O3. Thus, it is concluded that divanadium trioxide does not cause respiratory tract irritation.

 

No carcinogenicity, no pneumoconiosis and no other signs indicative of allergic inflammation have been reported for workers manufacturing vanadium dioxide.Therefore, the local respiratory effects of V2O5 are not relevant for read-across to divanadium trioxide.

 

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 and 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: F&M: 0, 125, 250, 500, 1000, 2000 mg/L) - already completed

- 90 days with Harlan Sprague-Dawley rats and B6C3F1/N mice (dose: F&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

- 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 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 classification or non-classification

The currently available and reliable toxicity data on vanadium substances does not justify classification of divanadium trioxide for specific target organ toxicity - repeated exposure.