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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:
June 17, 2010 - June 19,2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Phenolphthalein
EC Number:
201-004-7
EC Name:
Phenolphthalein
Cas Number:
77-09-8
Molecular formula:
C20H14O4
IUPAC Name:
phenolphthalein
Test material form:
solid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: vitro skin model RHE by SkinEthic Laboratories

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the treatment interval, the inserts were removed immediately from the 24-well plate. Using a multi pipette the tissues were gently rinsed with a minimum volume of 25 mL PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/L
- Incubation time: 3 hours
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability is less or equal than 50%.
- The test substance is considered to be non-irritant to skin if the viability is greater than 50%.

ACCEPTABILITY OF THE ASSAY
Negative control (NC acceptance criteria: The NC data meet the acceptance criteria if the mean OD value of the 3 tissues is ≥ 1.2 at 570 nm according to the historical database. The Standard Deviation value is considered as valid if it is ≤ 18 %, according to the Performance Standards (ECVAM SIVS, 2007).Positive control (PO acceptance criteria: The PC data meet the acceptance criteria if the mean viability, expressed as % of the NC, is < 40 % and the Standard Deviation value is ≤ 18 %.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration: 5% in PBS
Duration of treatment / exposure:
42 min
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1st run
Value:
36
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
2.64%
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

The results obtained after treatment are given in the following table:

Dose Group Treatment
Interval
Optical Density
Tissue 1
Optical Density
Tissue 2
Optical Density
Tissue 3
Mean relative
Viability [%]
neg. control 42 min 1.361 1.200 1.298 100.0
pos. control 42 min 0.033 0.036 0.034 2.64
test item 42 min 0.446 0.451 0.493 36.00


Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the treatment interval thus ensuring the validity of the test system.

After treatment with the negative control the absorbance values reached the required acceptability criterion of an OD >1.2 for the treatment interval thus showing the quality of the tissues.

The mean relative tissue viability after treatment with the test item was significantly decreased after treatment (mean relative viability = 36.00 %). Therefore, the test item is considered to possess an potential for skin irritation.

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Under the experimental conditions reported, the test item is irritant to skin.
Executive summary:

A study according study according B.46 of Council Regulation (EC) No. 761/2009 was performed to assess the irritation potential of the test item by means of the Human Skin Model Test. The test consisted of a topical exposure of the test item to a human reconstructed model followed by a cell viability test. Cell viability was measured by dehydrogenase conversion of MTT into a blue formazan salt, that was quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict skin irritation potential.

Triplicates of the human skin model RHE were treated either with the test item, the negative or the positive control for 42 minutes. 16 µL of either the negative control (PBS-buffer) or the positive control (5 % Sodiumdodecylsulphat-solution) were applied to each tissue. Before adding the test item, 10 µL of deionised water was spread to the epidermis surface to improve further contact between the test item and the epidermis. Afterwards, 16 mg of the test item were applied to each tissue.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the treatment interval thus ensuring the validity of the test system. After treatment with the negative control the absorbance values reached the required acceptability criterion of mean OD > 1.2 for the treatment interval thus showing the quality of the tissues. The tissue viability after treatment with the test item was significantly decreased (mean viability: 36.00 %). Therefore, the test item is considered to possess an irritant potential.

Under the experimental conditions reported, the test item is irritant to skin.