Sodium metavanadate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-11-07 to 2012-11-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010-07-22

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2009

Deviations:

no

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

Sodium metavanadate

EC Number:

237-272-7

EC Name:

Sodium metavanadate

Cas Number:

13718-26-8

Molecular formula:

NaVO3

IUPAC Name:

Sodium metavanadate

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material: sodium metavanadate
- Physical state: white powder

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)

In vitro test system

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 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 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 were applied to each tissue replicate, wetted with the 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

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

72.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no 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%).

Any other information on results incl. tables

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	1.674	1.529	1.647	1.617	72.2

* 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.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not irritating to the skin.
According to the Regulation (EC) No 1272/2008 and subsequent adaptations, the test item is not irritating to the skin.