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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-06-07 to 2017-06-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015-07-28
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009-07-23
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) for use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200).
Version / remarks:
2015-06-29
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2015-09-14

Test material

Constituent 1
Chemical structure
Reference substance name:
Aluminium vanadium tetraoxide
EC Number:
815-205-8
Cas Number:
13530-56-8
Molecular formula:
AlVO4
IUPAC Name:
Aluminium vanadium tetraoxide
Test material form:
solid: particulate/powder
Details on test material:
- State of aggregation: greenish brown powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: store at room temperature. Keep container tightly sealed. Protect from humidity and water.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: normal, human-derived epidermal keratinocytes
Cell source:
other: humans
Source strain:
other: not applicable
Details on animal used as source of test system:
not applicable
Justification for test system used:
In an international 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
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ skin model (source: MatTek Corporation, 82105 Bratislava, Slovakia)
- Tissue lot number: 25825
- Delivery date: 2017-06-27

TEMPERATURE USED FOR TEST SYSTEM
- Temperature of pre-incubation: 37 ± 1.5 °C (23 hours)
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 minutes, room temperature for 25 minutes
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
After the end of the treatment interval the tissues were 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 at least three times. Afterwards the inserts were once again rinsed with DPBS. The tissues were then transferred into plates with assay medium. Tissues were incubated for nearly 23 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. After incubation, the medium was changed (pre-warmed fresh medium). Thereafter tissues were incubated for another 18 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The complete incubation time was approx. 41 hours.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL (300 µL/ well)
- Incubation time with MTT: 3 hours
- Extraction of Formazan: after the incubation period, the tissues were rinsed three times with DPBS. The tissues were transferred into new plates containing extractant solution (isopropanol) in each well so that the tissues were covered completely and the plate was sealed to inhibit the evaporation of isopropanol. The formazan salt was extracted for 2.5 hours while shaking at room temperature.
After the extraction 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 and 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 after the 15 minutes exposure were transferred into a 96-well flat bottom microtiter plate. The optical density was read in a microplate reader. Mean values were calculated from the 3 wells per tissue.
- Spectrophotometer: Versamax® Molecular Devices
- Wavelength: 570 nm

TEST FOR COLOUR INTERFERENCE
Before the test started, a functional check for colour interference was performed. 25 ± 2 mg of the test item were added to deionised water. The mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 60 minutes. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated.
The test item dyed the water (brown), therefore, an additional test with viable tissues (but without MTT addition) had to be performed to determine a correction factor for calculating the true viability in the main experiment.
To check the tissue-binding of a coloured test item, two viable tissues were exposed to 25 ± 2 mg of the test item. In parallel, two tissues were exposed to DPBS (negative control). All procedures were followed as described for the MTT assay used in the main experiment, except that the tissue were incubated for 3 hours in culture media without MTT (37 ± 1.5°C, 5 ± 0.5% CO2,) instead of incubating in media containing MTT. After the 3 hour incubation, the tissues were rinsed and the tissues were extracted using isopropanol and the optical density (OD) at 570 nm was measured.
Since the tissues treated by the coloured test item (or test item) had a mean OD between 5% and 30% of the negative control tissue (treated with DPBS), the real MTT OD (unaffected by the interference with the coloured test items) was calculated using the following formula:
OD = ODcolored tissue (MTT assay) – ODcolored tissue (no MTT assay)

TEST FOR DIRECT MTT REDUCTION
The test item was evaluated for its potential to interfere with MTT assay. To test if a test item directly reduces MTT, 25 ± 2 mg of the test item were added to 1 mL of the MTT-solution (1 mg/mL) and was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) for 60 minutes. Untreated MTT medium was used as control.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tissues pass analysis for tissue viability
- Barrier function: tissues pass analysis for tissue functionality
- Morphology: presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as well as absence of HIV1- virus, Hepatitis B virus, and Hepatitis C virus
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA
The mean optical density (OD) of the three negative control tissues was calculated after blank correction. 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 is calculated according to the following formula: relative viability(%) = (mean OD test item or positive control/ mean OD 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 or equal to 50% of the negative control, the test item needs to be classified and labelled 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): approx. 25 mg (~ 39 mg/cm²) of the test item, wetted with vehicle

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

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of a 5% sodium lauryl sulfate (SLS) solution
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
approx. 41 hours
Number of replicates:
triplicates

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
26.6
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Colour interference with MTT: the test item dyed the water (brown), therefore, an additional test with viable tissues (without MTT addition) had to be performed to determine a correction factor for calculating the true viability in the main experiment.
- Direct-MTT reduction: the MTT solution did not turn blue/purple, the test item was not considered to reduce MTT and an additional functional test with freeze-killed tissues did not have to be performed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: after treatment with the negative control, the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval.
- Acceptance criteria met for positive control: treatment with the positive control induced a decrease in the relative absorbance compared with the negative control to 3.3 % (acceptability criterion: positive control is ≤ 20 %).
- Acceptance criteria met for variability between replicate measurements: the relative standard deviations between the % variability values of the test item, the positive and negative controls in the main test were below 5 % (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: < 18%).
Please refer to the field "Any other information on results incl. tables" below.

Any other information on results incl. tables

HISTORICAL DATA

Positive Control

Negative Control [OD570]

Mean Viability

4.37 %

Mean Absorption

1.74

Rel. Standard Deviation

21.60 %

Rel. Standard Deviation

9.40 %

Range of Viabilities

2.20 % - 6.78 %

Range of Absorbance

1.34 – 2.00

Mean Absorption

0.08

 

Rel. Standard Deviation

20.12 %

Range of Absorbance

0.03 - 0.11

Data of 103 studies performed from July 2015 until March 2017

Table 1: Results after treatment with aluminium vanadium tetraoxide and the controls

Dose Group

Tissue No.

Absor-bance 570 nm
Well 1

Absor-bance 570 nm
Well 2

Absor-bance 570 nm
Well 3

Mean Absor-bance of 3 Wells

Mean-Absor-bance

of three Wells blank

corrected

Mean

Absor-bance

of 3 Tissues

Mean Rel. Ab-sorbance

[%]*

Corrected Mean Rel. Absorbance [%]**

Blank

 

0.038

0.038

0.037

0.037

 

 

 

Negative Control

1

1.708

1.718

1.740

1.722

1.684

1.670

100.0

 

2

1.804

1.757

1.767

1.776

1.738

3

1.659

1.606

1.611

1.625

1.588

Positive Control

1

0.090

0.095

0.094

0.093

0.055

0.055

3.3

2

0.093

0.093

0.091

0.092

0.055

3

0.092

0.093

0.093

0.093

0.055

Test Item

1

0.527

0.507

0.505

0.513

0.476

0.445

27.3

26.6

2

0.482

0.474

0.472

0.476

0.438

3

0.499

0.486

0.484

0.489

0.452

Negative Controladd. viable

1

0.043

0.043

0.043

0.043

0.005

0.007

0.4

 

2

0.047

0.048

0.046

0.047

0.009

Test Itemadd. viable

1

0.047

0.047

0.047

0.047

0.010

0.010

0.6

2

0.048

0.049

0.048

0.049

0.011

*           Mean relative absorbance [rounded values]: [(100 x (mean absorbance test item/positive control /negative control)]/(mean absorbance negative control)

**                  Mean relative absorbance [rounded values]: [100 x (mean absorbance test item - mean absorbance test item add viable)]/(mean absorbance negative control)

               

Applicant's summary and conclusion

Interpretation of results:
other: may meet classification criteria for skin irritation or corrosion
Conclusions:
Based on the in vitro skin irritation test (OECD 439), aluminium vanadium tetraoxide is irritating to the skin. According to Regulation (EC) No 1272/2008 and its subsequent regulations, aluminium vanadium tetraoxide may meet classification criteria for skin irritation/corrosion.

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