Phenolphthalein

Skin irritation / corrosion

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 guideline

Test guideline 1

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 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Phenolphthalein

EC Number:

201-004-7

EC Name:

Phenolphthalein

Cas Number:

77-09-8

Molecular formula:

C20H14O4

IUPAC Name:

3,3-bis(4-hydroxyphenyl)-1,3-dihydro-2-benzofuran-1-one

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.