Vanadium oxide sulphate

Endpoint summary

Description of key information

Skin corrosion and Serious eye damage:

Vanadium oxide sulphate tested irritating to the skin in in vitro test systems according to OECD 439 and OECD 435. Regarding eye irritation, results of the in vitro test OECD 437 are inconclusive, i.e. no stand-alone predictions can be made. Nevertheless, vanadium oxide sulphate pentahydrate is a strong acid (pH< 2) and meets the classification criteria of Regulation (EC) No 1272/2008 for Skin Corr. 1 and Serious eye damage (H314: Causes severe skin burns and eye damage.)

Respiratory irritation:

Vanadium oxide sulphate is already classified for corrosivity and the related risk management measures are implemented.

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:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012-11-07 to 2012-11-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

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

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature (further detailed: Harlan CCR SOP SUBST.doc)

Test system:

human skin model

Source species:

human

Cell type:

other: normal, human-derived epidermal keratinocytes

Cell source:

other: not specified

Source strain:

not specified

Details on animal used as source of test system:

not applicable

Justification for test system used:

In a prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.

Vehicle:

other: Dulbecco's Phosphate Buffered Saline (DPBS)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm TM tissues (purchased from MatTek Corporation)
- Tissue lot number: 16852
- Delivery date: 2012-11-06
- Date of initiation of testing: 2012-11-07

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C (incubator; duration: 35 minutes) followed by placing the plates into a sterile hood (duration: 25 minutes)(total exposure duration: 60 minutes)
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
At the end of the treatment interval the inserts were removed from the plate and tissues were rinsed with DPBS in order to remove any residual test material. After the rinsing, the inserts were submerged in DPBS. Afterwards the inserts were again rinsed with DPBS. The tissues were then transferred into plates with fresh assay medium. The surface of the tissues was dried using sterile cotton tipped swaps. Tissues were incubated for about 23 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2.
New plates (or lower row of the same plates) were filled with 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 DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL
- Incubation time with MTT: 3 hours
- Extraction of formazan: after the incubation period, the MTT solution was aspirated from the wells, and the wells were rinsed with DPBS. Inserts were transferred onto new plates. The inserts were immersed into extractant solution by pipetting 2 mL isopropanol in each insert. The tissues were completely covered from both sides. The 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 to allow the extract to run into the well from which the insert was taken and the insert was discarded. The plates were shaken 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 was determined with a spectrophotometer. Mean values were calculated from the 3 wells per tissue.
- Spectrophotometer: Versamax® Molecular Devices
- Wavelength: 570 nm
- Filter bandwidth: 1 nm

TEST FOR DIRECT MTT REDUCTION
To test for the ability of the test item to directly reduce MTT approx. 25 mg of the test item were added to 1 mL of MTT solution 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.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function: tissues pass analysis for tissue functionality
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as well as absence of HIV1- virus, Hepatitis B virus, Hepatitis C virus
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA
The mean optical density 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 formula: 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 less than or equal to 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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approximately 25 mg of the test item was applied to each tissue replicate, wetted with vehicle

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 μL of DPBS

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

about 41 hours

Number of replicates:

Test item: triplicates
Negative control: triplicates
Positive control: triplicates

Irritation / corrosion parameter:

% tissue viability

Value:

30.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: 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.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: after treatment with the negative control, the absorbance values (2.117, 2.423, and 2.175, respectively) were well above the required acceptability criterion of mean OD ≥ 1.0 and ≤ 2.5 for the 60 minutes treatment interval.
- Acceptance criteria met for positive control: treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 3.8% (acceptability criterion: positive control is ≤ 20 %).
- Acceptance criteria met for variability between replicate measurements: 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%).

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 viabilities	1.423 – 2.651

Table 1: Results after treatment with vanadium oxide sulphate pentahydrate 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	0.685	0.578	0.763	0.676	30.2

* 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.

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, according to the current in vitro skin irritation test (OECD 439), vanadium oxide sulphate pentahydrate is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), i.e. vanadium oxide sulphate pentahydrate is irritating or corrosive to the skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-02-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

adopted 2009-09-07

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

, 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Details on test animals or tissues and environmental conditions:

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

Vehicle:

other: 0.9% (w/v) NaCl in deionised water

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
A 20% (w/v) solution of the test item in the vehicle was prepared using ultrasonic technique.

Duration of treatment / exposure:

240 ± 15 minutes

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

Test item: 3 corneas
Negative control: 3 corneas
Positive control: 3 corneas

Details on study design:

COLLECTION OF BOVINE EYES
Freshly isolated bovine eyes from at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were transported to the laboratory in Hank’s Ballanced Salt Solution (HBSS) supplemented with streptomycin / penicillin at ambient temperature. The corneae were isolated on the same day after delivery of the eyes, inserted in pre-cooled preservation medium composed of Medium 199 (© Biochrom) supplemented with L-glutamine, Na-bicarbonate and Taurine, and stored in the refrigerator at 2 – 8 °C until the following day. Shortly before use, Dextran was added to the medium.

PREPARATION OF CORNEAE
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors.
Each isolated cornea was mounted in a specially designed cornea holder as described in OECD guideline 437, annex III, that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. After the anterior part of the holder was positioned on top of the cornea and fixed in place with screws, both compartments of the holder were filled with complete medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure that no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.
At the end of the incubation period, the basal opacity was determined (t0). Each cornea with a value of the basal opacity > 7 was discarded and not used in the test.

OUTLINE OF STUDY
Complete medium was completely removed from the anterior compartment and replaced by the test item, positive control (10% (w/v) Benzalkonium chloride (Sigma, 89555 Steinheim, Germany, lot no. 036K0208) in 0.9% (w/v) NaCl solution in deionised water (saline, produced in-house, lot no. 180113)) or negative control (0.9% (w/v) NaCl in deionised water (produced in-house, lot no. 180113)).
The anterior compartment received the test item solution or negative or positive control at a volume of 0.75 mL each on the surface of the corneae.
The test item, positive control and negative control were tested in triplicate.
The anterior compartment was then plugged again. The corneae were turned into a horizontal position and slightly rotated to ensure uniform covering of the corneae with the test or control items and were incubated in a water-bath in horizontal position at 32 ± 1 °C for 240 minutes.
After the incubation, the test item or control items, respectively, were rinsed off from the application side with 0.9% (w/v) NaCl in deionised water at least three times or until no visual evidence of the test substance was observed. Fresh cMEM was added into the anterior compartment and opacity was measured (t240).
In the second step of the assay, permeability of the cornea was determined. 1 mL of a Na-fluorescein solution, 0.5 % (w/v) dissolved in HBSS (Hank’s buffered salt solution), was placed in the anterior compartment, replacing the cMEM. Corneae were incubated again in a horizontal position for an additional 90 minutes at 32 ± 1 °C in the water-bath. The optical density of an aliquot of the mixed complete medium from the posterior chamber was measured spectrophotometrically at 490 nm (OD490).

CRITERIA FOR DETERMINATION OF A VALID TEST
According to the OECD 437 protocol, the test its considered acceptable if the positive control gives an In Vitro Irritation Score (IVIS) that falls within two standard deviations of the current historical mean. The negative or solvent/vehicle control responses should result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control. Accordingly and based on historical control data, the test was acceptable if the in vitro irritation score of the positive control was ≥ 30 and the in vitro irritation score of the negative control was ≤ 3.

EVALUATION OF RESULTS
- Opacity: the change of opacity value of each treated cornea or positive and negative control corneae was calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
The average change in opacity of the negative control corneae was calculated and this value was subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
- Permeability: the corrected OD490 value of each cornea treated with positive control and test item was calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IN VITRO IRRITATION SCORE CALCULATION
The following formula was used to determine the in vitro irritation score of the negative control:
In vitro Irritation Score = opacity value + (15 x OD490 value)
The following formula was used to determine the in vitro irritation score of the positive control and the test item:
In vitro Irritation Score = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The in vitro irritation score was calculated for each individual treatment and positive control cornea. The mean in vitro irritation score irritation value of each treated group was calculated from the individual in vitro irritation score values.
Depending on the score obtained, the test item was classified into the following category according to OECD guideline 437 (table 1 in the field "Any other information on materials and methods incl. tables" below).

Irritation parameter:

in vitro irritation score

Value:

23.67

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not corrosive / not severely irritating to the eye

Other effects / acceptance of results:

Relative to the negative control, exposure of the test item vanadium oxide sulphate pentahydrate to the corneae did not induce an increase of the corneal permeability, but an increase of the opacity values occurred. The calculated mean in vitro irritation score was 23.67 (threshold for corrosivity / severe irritancy: ≥ 55.1). According to OECD guideline 437, the test item is not classified as corrosive / severe irritant to the eye.

Table 1: Results after 240 Minutes Incubation Time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		In vitro Irritation Score	Mean in vitro irritation score   ± Standard deviation	Proposed in vitro Irritation Scale
		Mean		Mean
Negative Control	5	2.00	0.052	0.055	5.78	2.83 ± 2.60	Non corrosive / non severe irritant
	1		0.055		1.83
	0		0.059		0.89
Positive Control	146.00*		0.001*		146.01*	213.03 ± 90.54	Corrosive / severe irritant
	316.00*		0.002*		316.03*
	177.00*		0.003*		177.04*
Vanadium oxide sulphate pentahydrate	10.00*		- 0.009*/**		10.00*	23.67 ± 13.05	Non corrosive / non severe irritant
	25.00*		-0.009*/**		25.00*
	36.00*		- 0.011*/**		36.00*

 * Corrected values (subtraction of mean opacity/permeability of negative control from measured opacity/permeability of each replicate, respectively)

** Value was set to “0” for the calculation of the irritation score

- With the negative control (0.9% (w/v) NaCl in deionised water) neither an increase of opacity nor permeability of the corneae could be observed (mean in vitro irritation score 2.83). The score is well within the concurrent negative control range, i.e. within two standard deviations of the current historical mean.

- The positive control (10% (w/v) Benzalkonium chloride in 0.9% (w/v) NaCl in deionised water) induced clear opacity of the corneae (mean in vitro irritation score 213.03) corresponding to a classification as corrosive /severe irritant to the eye (CLP/EPA/GHS (Cat 1)). The score is well within the concurrent positive control range, i.e. within two standard deviations of the current historical mean.

- Thus, based on historical positive and negative control data, this test meets the acceptability criteria in accordance with OECD 437.

Table 2: Historical data

	Positive Control	Negative Control
Mean in vitro Irritation Score	176.71	1.78
Standard Deviation	42.65	0.75
Range of in vitro Irritation Scores	99.4 - 292.3	0.41 - 2.99

Values of 138 studies with solid test items performed between February 2007 and November 2012

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, according to the current in vitro BCOP test (OECD 437), no stand-alone prediction of the ocular irritation potential of vanadium oxide sulphate pentahydrate can be made. Nevertheless, vanadium oxide sulphate pentahydrate is a strong acid (pH< 2) and meets the classification criteria of Regulation (EC) No 1272/2008 for Skin Corr. 1A and Serious eye damage (H314: Causes severe skin burns and eye damage).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In the in vitro skin corrosion test (OECD 435), vanadium oxide sulphate pentahydrate applied as is (not moistened) did not test as corrosive to the skin. The substance is known to show strong acidic properties in aqueous solutions, thus any testing in the absence of water does not reflect the corrosive properties of the substance correctly. Consequently, this test is considered not reliable and disregarded for the hazard assessment.

Vanadium oxide sulphate pentahydrate is a strong acid (pH< 2, see section 4.8) and meets the classification criteria of Regulation (EC) No 1272/2008 for Skin Corr. 1 and Serious eye damage (H314: Causes severe skin burns and eye damage).

Justification for classification or non-classification

Skin corrosion and Serious eye damage:

Vanadium oxide sulphate pentahydrate is a strong acid (pH< 2) and meets the classification criteria of Regulation (EC) No 1272/2008 for Skin Corr. 1 and Serious eye damage (H314: Causes severe skin burns and eye damage.)

Respiratory irritation:

Vanadium oxide sulphate is already classified for corrosivity and the related risk management measures are implemented.