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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:
weight of evidence
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2010-07-22
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009
Deviations:
no
Qualifier:
according to
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. certificate)
Remarks:
signed 2009-03-30

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Vanadium oxide sulphate pentahydrate
- Trivial name: vanadyl sulphate
- Physical state: blue crystals

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 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

Results and discussion

In vitro

Results
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%).

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 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 (absorbancetest item)/(absorbancenegative 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:
Category 2 (irritant)
Conclusions:
The test item is irritating to the skin.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item is irritating to the skin (Category 2, H315: Causes skin irritation).