Potassium vanadium trioxide

Endpoint summary

Description of key information

An acute in vitro skin irritation / corrosion test according to OECD 439 (Heppenheimer, 2012) was performed with the structural analogue sodium metavanadate, and results indicate that it is not irritant to skin.

An acute in vivo eye irritation / corrosion test according to OECD 405 (Hansen, 2013) was performed with the structural analogue sodium metavanadate, and results indicate that it is irritant to eyes. Beforehand, an in vitro eye corrosion test had been conducted according to OECD 437 (Heppenheimer, 2013) to assure that the substance is not corrosive to the eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

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

Adequacy of study:

key study

Study period:

2012-11-07 to 2012-11-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted 2010-07-22

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

, 2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: In vitro EpiDerm TM Skin irritation Test (EPI-200-SIT) for use with MatTek Corporation's Reconstructed Human Epidermal Model EpiDerm (EPI-200), Rev. 1/19/2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Details on test animals or test system and environmental conditions:

Not applicable - Since this is an in vitro study there is no information on test animals.

Vehicle:

other: Dulbecco's Phosphate Buffered Saline (DPBS)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approximately 25 mg (using a weighing spoon) of the test item were applied to each tissue replicate, wetted with the vehicle, and spread to cover the tissue.

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 μL of Dulbecco's Phosphate Buffered Saline

Duration of treatment / exposure:

60 minutes

Observation period:

not applicable

Number of animals:

not applicable

Details on study design:

CELL CULTURE
Epi-200 SIT kits (Lot No. 16852) and MTT-100 assays diluent were purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm ∅).
EpiDerm™ tissues were shipped at 4 °C on medium-supplemented agarose gels in a 24-well plate and reached the laboratory on November 06, 2012. On day of receipt the preincubation phase of the EpiDerm™ tissues started.
The RhE model supplier ensured and demonstrated that each batch of the RhE model used met defined production release criteria, e.g. viability, barrier function and morphology (please refer to "Attached background material" below).

TEST FOR DIRECT MTT REDUCTION
For a correct interpretation of results it was necessary to assess the ability of the test item to directly reduce MTT. To test for this ability approximately 25 mg of the test item were added to 1 mL of MTT solution (using a weighing spoon) and the mixture was incubated in the dark at room temperature for 60 minutes. Untreated MTT medium was used as control. If the colour of the MTT would turn blue/purple, the test item is assumed to have reduced MTT.
The optical evaluation of the MTT-reducing capacity of the test item after 1-hour incubation with the MTT-reagent indicated that it did not turn blue. Therefore, the test item did not reduce MTT directly and a functional test with freeze-killed tissue was not deemed necessary.

TREATMENT
- Pre-warming of EpiDerm™ Tissues:
One day prior to the performance, the plastic bag containing the 24-well plate with epidermal tissues was opened under sterile conditions. Under a airflow using forceps, the gauze was removed and the inserts were taken out. Any remaining agarose that adheres to the outer sides of the inserts was removed by gentle blotting on the sterile filter paper or gauze, and the tissues were placed in the empty, sterile 6-well plate. Prior to the exposure of the test item and of the controls the EpiDerm™ tissues were inspected for quality: If necessary, it was taken care, that
1. air bubbles between agarose and insert were not > 30% of the total surface,
2. liquid on top of the insert was removed with steriles cotton tips,
3. if again moisture is observed on top of the inserts after the pre-incubation or in case of visible defects, the respective skin models were discarded.
0.9 mL of the DMEM based, serum-free assay medium (20 – 25 °C) was pipetted into each of the wells of the sterile 6-well plates. The inserts with the EpiDerm™ tissues were placed in the upper wells, and were pre-incubated for 60 minutes in the incubator (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH). Afterwards, the inserts were transferred from the upper wells into the lower wells of the 6-well plates, and, the preincubation was continued for further 24 hours (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH).
- Treatment: after pre-incubation of EpiDerm™ tissues was completed, the negative and positive control and the test item were added into the insert atop the corresponding EpiDerm™ triplicate tissues. The 6-well plates were placed into the incubator for 35 minutes at 37 ± 1.5 °C, 5 ± 0.5 % CO2, 95 ± 5% RH. Thereafter, plates were removed from the incubator and placed into the sterile hood until the exposure period of 60 minutes was completed.
At the end of the treatment interval the inserts were removed immediately from the 6-well plate and tissues were gently rinsed with DPBS for at least 15 times in order to remove any residual test material. After the rinsing, the inserts were submerged in DPBS for at least three times. Afterwards the inserts were again rinsed with DPBS from the inside and the outside. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with sterile blotting paper. The tissues were then transferred into new 6-well plates with 0.9 mL of fresh assay medium in the upper row. The surface of the tissues was carefully dried using sterile cotton tipped swaps. Tissues were incubated for about 23 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2.
New 6-well plates (or lower row of the same plates) were filled with 0.9 mL of fresh assay medium, and the inserts were transferred onto the new plates. The wells were incubated for nearly 18 hours post-incubation at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The complete incubation period was about 41 hours.

MTT ASSAY
Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethylthiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues (Faller, C., Bracher, M., Dami, N., Roguet, R., 2002. Predictive ability of reconstructed human epidermis equivalents for assessment of skin irritation of cosmetics. Toxicology in vitro 16 (5), 557-552).
The percent reduction of cell viability in comparison of untreated negative controls is used to predict skin irritation potential (see OECD TG 439) and is used for the purpose of classification as irritating or non-irritating to skin according to chemical regulation (Regulation (EC) No 1272/2008 (EU CLP), UN GHS).
After the 42-hours incubation period was completed for all tissues and exposure groups, culture inserts were transferred from the holding plates to the MTT-plates containing 300 µL MTT solution. After a 3-hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2), the MTT solution was aspirated from the wells, and the wells were rinsed three times with DPBS. Inserts were transferred onto new 24-well plates. The inserts were immersed into extractant solution by gently pipetting 2 mL extractant solution (isopropanol) in each insert. The level rose above the upper edge of the insert, thus tissues were completely covered from both sides. The 24-well plate was sealed to inhibit the isopropanol evaporation. The formazan salt was extracted for about 69 hours at room temperature.
After the extraction period was completed, the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken. Afterwards the insert was discarded. The 24-well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour.
Per each tissue, 3 × 200 μL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate from the 15 minutes exposure. The optical density (OD) was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany) with a 570 ± 1 nm filter. Mean values were calculated from the 3 wells per tissue.

EVALUATION OF RESULTS
The mean optical density (OD) of the three negative control tissues was calculated. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability was calculated according to the following formulas:
Relative viability (%) = (OD test item/ OD mean of negative control) X 100
For the test item and the positive control, the mean relative viability ± relative standard deviation of the three individual tissues was calculated and used for classification according to the following prediction model:
if the mean relative tissue viability of three individual tissues is reduced below 50% of the negative control, the test item needs to be classified and labeled for its skin irritation potential: Category 2 – irritant, H315 according to Regulation (EC) No 1272/2008 (Xi, R38 – irritating to skin according to Directive 67/548/EEC).

ACCEPTABILITY OF THE ASSAY
Concurrent negative controls (NC) and positive controls (PC) were used in each run to demonstrate that viability (with the NC), barrier function and resulting tissue sensitivity (with the PC) of the tissues were within a defined historical acceptance range.
Criterion 1 (negative control): the absolute OD (at 570 nm) of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD570 of the negative control tissues is ≥ 1.0 and ≤ 2.5 in accordance with the OECD 439 guideline.
Criterion 2 (positive control): an assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 20% (threshold established at the laboratory).
Criterion 3: Standard deviation: the SD of 3 identical tissue replicates should be < 18%.
OD values should not be below historically established boundaries.
According to the OECD 439 guideline, the acceptance limit of the ET50 should be between 4.8 hours and 8.7 hours after treatment with 1% Triton X-100 (QC batch release criteria). The respective tissue passed this functionality test (please refer to "Attached background material" below).

Irritation / corrosion parameter:

other: other: relative viability (%)

Value:

72.2

Remarks on result:

other:

Remarks:

Basis: mean. Time point: after 60 min. incubation. Reversibility: no data. (migrated information)

Irritant / corrosive response data:

Compared to the relative absorbance value of the negative control, the mean relative absorbance value decreased to 72.2% after exposure of the skin tissues to the test item sodium metavanadate. This value is well above the viability threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess a skin irritating potential.

HISTORICAL DATA

Positive Control		Negative Control
Number of Studies	67	Number of Studies	67
Period	May 2010 – November 2012	Period	May 2010 – November 2012
Mean Viability	6.6%	Mean Absorption	1.717
Standard Deviation	2.1%	Standard Deviation	0.274
Range of Viabilities	4% - 9.4%	Range of Vabilities	1.423 – 2.651

Table 1: Results after treatment with sodium metavanadate and the controls

Dose group	Treatment interval	Absorbance 570 nm Tissue 1*	Absorbance 570 nm Tissue 2*	Absorbance 570 nm Tissue 3*	Mean Absor-bance of 3 Tissues	Mean Rel. Absorbance [% of Negative Control]**
Negative control	60 min	2.117	2.423	2.175	2.239	100.0
Positive control	60 min	0.091	0.088	0.077	0.085	3.8***
Test item	60 min	0.844	0.835	0.740	0.806	36.0

* Mean of three replicate wells after blank correction

** relative absorbance [rounded values]: 100 x (absorbance_{test item})/(absorbance _{negative control})

*** The viability of the positive control is below the historical limit of 4.0%. Nevertheless, since the positive control induced a clear positive effect, and is only slightly below the historical limit, it can still be considered as valid.

- after treatment with the negative control, the absorbance values were well above the required acceptability criterion of mean OD ≥ 1.0 and ≤ 2.5 for the 60 minutes treatment interval, and thus assuring the quality of the tissues.

- treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 3.8%, and thus assuring the validity of the test system.

- the relative standard deviations of readings for tissue replicates of the test item, positive and negative controls were all below 18%, respectively (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: 18%), thus assuring the validity of the study.

The optical evaluation of the MTT-reducing capacity of the test item after 1-hour incubation with the MTT-reagent indicated that it did not turn blue and thus did not reduce MTT directly.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In this study and under the reported experimental conditions of the Human Skin Model Test, the test item sodium metavanadate is not irritant to skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

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

Adequacy of study:

key study

Study period:

2013-02-25 to 2013-03-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

adopted 2012-10-02

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-11-12

Species:

rabbit

Strain:

Himalayan

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Branch Löhndorf, 24601 Löhndorf/Post Wankendorf, Germany
- Age at study initiation: approximately 7 months
- Weight at study initiation: 2.50 - 2.85 kg
- Housing: for 8 hours following test item application, the animals were kept singly in restrainers which allowed free movement of the head but prevented a complete body turn, wiping of the eyes with the paws and excluded irritation of the eye by excrements and urine. During the acclimatisation period and after the 8-hour period in restrainers, the animals were kept singly in cages with dimensions of 380 mm x 425 mm x 600 mm (manufacturer: Dipl.Ing. W. EHRET GmbH, 16352 Schönwalde, Germany).
- Diet (ad libitum; before and after the exposure period): commercial diet, ssniff® K-H V2333 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum; before and after exposure period): tap water
- Acclimation period: at least 20 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature: 20°C ± 3°C (maximum range)
- Relative humidity: 30% - 70% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg of the test item were administered into one eye each of three animals. The test item was placed into the conjunctival sac of the right eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second in order to prevent loss of the material. The left eye, which remained untreated, served as a control.

Duration of treatment / exposure:

One hour

Observation period (in vivo):

Prior to the administration and 1, 24, 48, 72 hours and 4 to 6 days after the administration

Number of animals or in vitro replicates:

3 female rabbits

Details on study design:

USE OF TOPICAL ANAESTHETICS AND SYSTEMIC ANALGESICS
Sixty minutes prior to test item administration, 0.01 mg Buprenovet®/kg b.w. were administered by subcutaneous injection to all animals to provide a therapeutic level of systemic analgesia to avoid or minimize pain and distress.
Five minutes prior to the test item administration, one or two drops of Ophtocain®, a topical anaesthetic, was applied to each eye of all animals, to the right eye, in which the test item was to be applied, and to the left eye, which served as control.
In addition, 8 hours after administration of the test item all animals were treated with 0.01 mg Buprenovet®/kg b.w. in conjunction with 0.5 mg Metacam®/kg b.w., subcutaneously.

INITIAL TEST AND CONFIRMATORY TEST
The test was performed initially using one animal. As no corrosive or severe irritant effects were observed in this animal, 2 further animals were employed 24 hours after start of the initial test.

REMOVAL OF TEST SUBSTANCE
- Washing: each eye was rinsed with 20 mL of 0.9% aqueous NaCl solution.
- Time after start of exposure: one hour after instillation

SCORING SYSTEM: according to the Draize scale
Any further lesions are listed.

TOOL USED TO ASSESS SCORE: the eyes were examined ophthalmoscopically with a slit lamp prior to the administration and 1, 24, 48, 72 hours and 4 to 6 days after the administration. The eye reactions were observed and registered.
24 hours after administration, fluorescein (Fluorescein SE Thilo drops (ALCON PHARMA GmbH, 79108 Freiburg, Germany)) was applied to the eyes before being examined to aid evaluation of the cornea for possible lesions.

OBSERVATIONS:
General criteria: body weight of all animals was measured at the beginning and at the end of the study. Behaviour and food consumption were monitored.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

3.33

Max. score:

4

Reversibility:

fully reversible within: 6 days

Remarks on result:

other: Corneal staining (3/4 to whole surface) was observed during the 24 hours fluorescein test.

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

2

Reversibility:

fully reversible within: 4 days

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

fully reversible within: 6 days

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

4

Max. score:

4

Reversibility:

fully reversible within: 6 days

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 5 days

Remarks on result:

other: Corneal staining (1/2 to 3/4 of the surface) was observed during the 24 hours fluorescein test.

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

2.67

Max. score:

3

Reversibility:

fully reversible within: 5 days

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

fully reversible within: 6 days

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 5 days

Remarks on result:

other: Corneal staining (3/4 to whole surface) was observed during the 24 hours fluorescein test.

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 5 days

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 4 days

Irritant / corrosive response data:

A single instillation of 100 mg Sodium metavanadate per animal into the conjunctival sac of the right eye of three rabbits caused the following changes:
Cornea opacity was observed in all animals:
- one animal: 24 and 72 hours (grade 3), 48 hours (grade 4), 4 and 5 days (grade 1) after instillation;
- second and third animal: 24 hours to 4 days (grade 1) after instillation.

The irises (grade 1) were observed in animal no. one 48 and 72 hours after instillation.

Conjunctivae redness was observed in all animals:
- one animal: 60 minutes and 4 to 5 days (grade 1), 24 to 72 hours (grade 3) after instillation;
- second animal: 60 minutes and 4 days (grade 1), 24 and 48 hours (grade 3), 72 hours (grade 2) after instillation;
- third animal: 60 minutes and 72 hours to 4 days (grade 1), 24 hours (grade 3), 48 hours (grade 2) after instillation.

Chemosis was observed in all animals:
- one animal: 60 minutes and 4 to 5 days (grade 1), 24 to 72 hours (grade 4) after instillation;
- second animal: 60 minutes and 4 days (grade 1), 24 hours (grade 4), 48 hours (grade 3), 72 hours (grade 2) after instillation;
- third animal: 24 hours (grade 3), 48 hours (grade 2), 72 hours (grade 1) after instillation.

Other effects:

There were not any systemic intolerance reactions concerning behaviour, body weight and food consumption.

Interpretation of results:

other: Category 2 (irritating to eyes)

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Sodium metavanadate oxide is irritating to the eyes.
According to the EC-Commission directive 67/548/EEC and its subsequent amendments, sodium metavanadate is classified as irritating to the eyes.
According to the EC-Regulation 1272/2008 and subsequent regulations, sodium metavanadate is classified in Category 2.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Read-across:

KVO3 and NaVO3 are vanadates with similar solubility potential, i.e. 124 g/L at pH 9 and 215 g/L at pH 8.8, respectively, and similar V content, i.e. 36.9% and 41.8%, respectively. Potassium and sodium are (i) ubiquitous in the environment (air, soil and water), (ii) key nutrients and intrinsically present in the human body due to a natural background in all dietary sources, and (iii) normal constituents of all body fluids and main blood minerals, and maintain electrolyte or acid-base (pH) balance as well as a proper fluid. Potassium and sodium belong to the ten most common elements in the human body, by mass ca. 0.4% and 0.2%, respectively, and functions in the body cover cellular transport, water regulation, blood pressure control, nerve conduction, muscular contraction as well as enzymatic reactions and other metabolic activities. In consequence, because of the intrinsic nature of sodium and potassium as endogenous physiological compounds and because of similar solubility characteristics of KVO3 and NaVO3, read-across of skin and eye irritation data from NaVO3 to KVO3 is justified.

Justification for selection of skin irritation / corrosion endpoint:

reliable in vitro GLP guideline study with structural analogue

Justification for selection of eye irritation endpoint:

reliable in vivo GLP guideline study with structural analogue

Effects on eye irritation: irritating

Justification for classification or non-classification

Skin irritation:

Potassium vanadium trioxide does not possess an irritation potential and does not require classification as skin irritant according to Regulation (EC) 1272/2008.

Eye irritation:

Potassium vanadium trioxide possesses an irritation potential and requires classification as eye irritant according to Regulation (EC) 1272/2008 (category 2).

Respiratory irritation:

Potassium vanadium trioxide is neither irritating/corrosive to skin nor corrosive to eyes. Only mild, reversible effects were observed in the in vivo eye irritation test (Hansen, 2013). Furthermore, local reversible or irreversible adverse health effects were not observed below lethal levels (i.e. no pathological findings) in the acute inhalation toxicity test (Leuschner, 1992). Hence, potassium vanadium trioxide does not possess an irritation potential in the respiratory tract and does not require classification as respiratory irritant according to Regulation (EC) 1272/2008.