Vanadium

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Vanadium carbide nitride (V(C,N))

IUPAC Name:

Vanadium carbide nitride (V(C,N))

Constituent 2

Reference substance name:

Vanadium carbide nitride

EC Number:

601-732-9

Cas Number:

12069-91-9

Molecular formula:

VCN

IUPAC Name:

Vanadium carbide nitride

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Vanadium Carbide Nitride
- Physical state: metallic grey solid
- Storage condition of test material: room temperature in a desiccator

Test animals

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

Administration / exposure

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

Doses / concentrations

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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

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

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

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.