Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-05-08 to 2014-06-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD Guideline 439 In vitro Skin Irritation: Reconstructed Human Epidermis Test method, adopted July 26, 2013
Deviations:
no
Qualifier:
according to
Guideline:
other: Council Regulation (EC) No. 761/2009, method B.46. In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test, adopted August 24, 2009
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
other: three-dimensional reconstructed human epidermis model EpiDermTM
Strain:
other: Not applicable
Details on test animals and environmental conditions:
The following Reconstructed Human Epidermis Model was used:
EpiDermTM (EPI-212-SIT, Lot no. 19642) MatTek In Vitro Life Science Laboratories, Bratislava, Slovak Republic.

Test system

Type of coverage:
other: The test substance was applied to the skin model to uniformly cover the skin surface.
Preparation of test site:
other: Not applicable as three-dimensional reconstructed human epidermis model EpiDermTM.
Vehicle:
unchanged (no vehicle)
Remarks:
But the epidermis surface was moistened with Dulbecco's phosphate buffered saline (D-PBS) before application to ensure good contact with the skin.
Controls:
other: Concurrent negative (Dulbecco's phospahe buffered sline) and positive control (SDS) each in triplicate, to demonstrate that viability (NC), barrier function and resulting issue sensitivity (PC) of the tissues are acceptance range.
Amount / concentration applied:
25 mg on the surface area of 0.63 cm².
Duration of treatment / exposure:
See "Details on study design"
Observation period:
See "Details on study design"
Number of animals:
3 samples were applied to the skin model
Details on study design:
1.1 General model conditions
Normal human keratinocytes were used to construct the epithelium. Multiple layers of viable epithelial cells (basal layer, stratum spinosum, stratum granulosum) were present under a functional stratum corneum. Stratum corneum was multilayered containing the essential lipid profile to produce a functional barrier with robustness to resist rapid penetration of the cytotoxic marker substance sodium dodecyl sulphate (SDS). The barrier function is assessed either by determination of the concentration at which a marker substance reduces the viability of the tissues by 50% (IC50) after a fixed exposure time, or by determination of the exposure time required to reduce cell viability by 50% (ET50) upon application of the marker substance at a specified, fixed concentration. The containment properties of the model prevented the passage of material around the stratum corneum to the viable
tissue, which would lead to poor modelling of skin exposure. The skin model was free of contamination by bacteria, viruses, mycoplasma, or fungi.

1.2 Functional model conditions
The preferred assay for determining the magnitude of viability was the MTT. The optical density (OD) of the extraction solvent alone were sufficiently small, i.e. OD < 0.1. The tissue treated with NC should exhibit stability in culture (provide similar viability measurements) for the duration of the test exposure period.

2. Quality controls (QC) of the model
The EpiDerm™ System was manufactured according to defined quality assurance procedures. All biological components of the epidermis and the culture medium were tested by manufacturer for viral, bacterial, fungal and mycoplasma contamination. The supplier determines the ET50 value following exposure to Triton-100 (1%) for each EpiDerm lot. The ET50 must fall within a range established based on a historical database of results.
The certificate of the EpiDermTM model has been attached to the study report.

3. Test for interference of chemicals with MTT assay
To identify the potential of the test item to interfere with MTT assay, the following check was performed:
25 mg test item was mixed with 300 μL sterile deionised water and incubated in the dark at 37°C, 5% CO2 and 95% humidity for 60 minutes. No discolorations were noted. Hence, no possible interacting with the MTT measurement had to be considered and no additional test had to be performed.

4. Administration of the test, negative and positive reference items
25 mg of test item were applied to the phosphate buffered saline moistened skin model with a surface area of 0.63 cm2 to uniformly cover the skin surface. A minimum of 25 mg or 25 μL substance applied per cm2 is required by the guidelines. Three replicate tissues were employed. At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS).
The whole exposure period for the used EpiDermTM skin model was 60 minutes.
The incubation conditions were 37°C, 5% CO2 and 95% humidity for the first 35 minutes followed by 25 minutes at room temperature under a sterile hood. Concurrent negative and positive controls were used, each in triplicate, to demonstrate that viability (NC), barrier function and resulting issue sensitivity (PC) of the tissues are within a defined historical acceptance range. The positive control item was 5% aqueous sodium dodecyl sulphate (SDS) and the negative control was D-PBS. 30 μL of negative and positive controls were used.

5. Cell viability measurements
The MTT conversion assay is a quantitative validated method which is used to measure cell viability. It is compatible with use in a three-dimensional tissue construct. The most important element of the test procedure was that viability measurements were not performed immediately after the exposure to the test item, but after a posttreatment incubation period of the rinsed tissues in fresh assay medium of 42 hours.
This period allows both for recovery from weakly irritant effects and for appearance of clear cytotoxic effects. Each skin sample was placed in an MTT assay solution of 1 mg/mL (37°C incubation temperature, 5% CO2, 95% humidity) for 3 hours. The precipitated blue formazan product was extracted using the extraction solution (isopropanol), and the concentration of the formazan was measured by determining the optical density (OD) at a wavelength of 570 nm in a spectrophotometer. The measurements were made for each of the three tissues in duplicate.

6. Assay acceptability criteria
Assay acceptance criterion 1: Negative control
The absolute OD of the negative control (NC) tissues (treated with sterile PBS buffer) in the MTT test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use.
The assay meets the acceptance criterion if the mean OD570 of the NC tissues is ≥ 1.0 and ≤ 2.5.
Assay acceptance criterion 2: Positive control
A 5% SDS (in H2O) solution was used as positive control (PC) and tested concurrently with the test chemicals. Concurrent means here the PC has to be
tested in each assay, but not more than one PC is required per testing day.
The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues is ≤ 20%.
Assay acceptance criterion 3: Standard deviation
Since in each test skin irritancy potential is predicted from the mean viability determined on 3 single tissues, the variability of tissue replicates should be acceptably low.
The assay meets the acceptance criterion if the standard deviation (SD) calculated from individual % tissue viabilities of the 3 identically treated replicates is < 18%.

7. Interpretation of results
The OD values obtained for each test sample were used to calculate mean percentage viability relative to the negative control, which was arbitrarily set at 100%. The cut-off mean percentage cell viability value that distinguishes irritant from non-classified test substances is given below:
The test item has to be considered to be irritant to skin in accordance with UN GHS category 2, if the tissue viability after exposure and post-treatment incubation is below or equal 50%.
The test item has to be non-irritant if the tissue viability after exposure and posttreatment incubation is higher than 50%.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of 3 experiments
Value:
103.1
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

The EpiDermTM model was employed.

Three tissues were used for each treatment and concurrent control groups. The optical density (OD) was determined by using the MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue) reduction assay and expressed as relative percentage of viability of the negative control-treated tissues.

L-Phenylalanine was applied topically as solid test item to the model skin surface, which was moistened with phosphate buffered saline. Dulbecco’s phosphate buffered saline (D-PBS) was used as the negative control. 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item.

The mea n viability of cells exposed to L-Phenylalanine was 103.1% of the negative controls and, hence, was well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of > 50%. L-Phenylalanine was considered to be non-cytotoxic and predicted to be nonirritant to skin. The mean optical density (OD) of the negative control of 3 tissues was 2.160 and was well within the acceptable range of ≥ 1.0 to ≤ 2.5. The viability of cells treated with the positive reference item, 5% SDS, was 3.1% of the negative control and fulfilled the acceptance criteria of < 20%. The standard deviation of all triplicates determined was below the limit of acceptance of 18%. Hence, all acceptance criteria were fulfilled. Conclusion: Under the present test conditions, L-Phenylalanine tested at an exposure time of 60 minutes and a 42-hour post-treatment incubation period, was non-cytotoxic and, hence, was considered to be non-irritant to skin in an experiment employing an artificial three-dimensional model of human skin.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
In a GLP gudeline study according to OECD 439 (three-dimensional reconstructed human epidermis model) L-phenylalanine did not show to be irritatting to the skin.