Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 14th to 28th, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
July 28th, 2015
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Tris(ethyl acetoacetato-O1',O3)aluminium
EC Number:
239-343-8
EC Name:
Tris(ethyl acetoacetato-O1',O3)aluminium
Cas Number:
15306-17-9
Molecular formula:
C18H27AlO9
IUPAC Name:
Aluminum, tris(ethyl 3-oxobutanoato-O1',O3)
Test material form:
solid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: adult donors
Details on animal used as source of test system:
SOURCE ANIMAL
- Source:Human adult donors.
Justification for test system used:
Test system used according to the OECD TG 439. The test system EPISKIN™ is a reconstructed human epidermis (RhE) model, which in its overall design (the use of human derived epidermis keratinocytes as cell source and use of representative tissue and cytoarchitecture) closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Episkin Small model- 0.38 cm^2
- Tissue batch number:16-EKIN-047
- Shipping date: Monday 21 November 2016
- Delivery date: Tuesday 22 November 2016. Note: at arrival ther test system was examinated and it resulted suitable for use (temperature indicator:pale grey; pH indicator: orange). Plates were opened under a sterile airflow and each insert, containing the epidermal tissue, was carefully taken out and placed in a 12-well plate in which each well had previously been filled with 2 ml/well SkinEthic Maintenance Medium. Culture plates were placed in the incubator at 37 °C, 5 % CO2 and saturated humidity for approximately 24 hours.

PRELIMINARY TESTS:
Before the Main Assay, a preliminary tests were carried out to evaluate the compatibility of
the test item with the test system.
- Direct-MTT reduction: 2 ml of MTT ready-to-use solution (0.3 mg/ml) was incubated with 20±2 mg of test item at 37 °C, 5% CO2 and saturated humidity for 3 hours, simulating test conditions. Observation of blue or purple appearance of the solution at the end of the incubation time was carried out.
- Colour interference with MTT: 20±2 mg of the test item was added to 180 µl of distilled water in at transparent tube and the resulting suspension mixed by using a vortex for 15 minutes. Colouring of the suspension at the end of the incubation time was evaluated by spectral analysis at 595 nm.

MAIN TEST
The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 ± 1 hour recovery period. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system, being this reaction an index of cell viability.

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: at the end of the exposure, each tissue was rinsed with approximately 25 ml of sterile D-PBS, filling and empting the tissue insert. The excess liquid was carefully removed and the sample transferred in new wells pre-filled with 2 ml/well of maintenance medium.
- Observable damage in the tissue due to washing: not observed

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 ml/well of MTT ready-to-use solution
- Incubation time: 3 hours at 37 °C, 5% CO2 and saturated humidity.At the end of the incubation period, tissues were placed on absorbent paper to dry. A total biopsy was carried out by means of a biopsy punch to allow biopsies of the same dimensions. The epidermis were separated from the collagen matrix and both placed in a microtube prefilled with 500 µl of acidic isopropanol. Tubes were preserved for approximately 3 days at 4 °C to allow formazan extraction. At the end of the extraction period, debris were eliminated by short centrifugation of the tubes (14000 rpm for 2 minutes) and aliquots of 200 µl from each sample were read in duplicate for their absorbance at 595 nm. Optical Density (OD) values were recorded. Six aliquots (200µl) of acidic isopropanol were analysed and used as blank. In order to ensure the spectrophotometer linear range , an MTT formazan calibration curve was performed. A deviation occurred during the study but this deviation didn't influence the result OR the reliability of the study: internal Quality Control solutions of MTT formazan were analysed the day of the experimental plate reading but they gave OD values around 60% of the expected values, so they were regarded as invalid. For this reason, another MTT formazan curve was performed the same week of the study. The values obtained with this second analysis were acceptable and comparable to the previous MTT formazan curve, thus confirming the adeguacy of the spectrophotometer and the validity of the data obtained for the experiment.
- Wavelength:the absorbance was evaluated at 595 nm.

NUMBER OF REPLICATE TISSUES: 3 replicates for negative control, positive control and test item and two replicates for test item without MTT. Note:the test item without MTT is Non Specific Colouring potential (NSC living) using to evaluate the potential colouring of the test item.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-corrosive and non-irritant to skin if the viability after an exposure period of 15 minutes followed by 42 ± 1 hour recovery period is greater than 50%.

ACCEPTANCE CRITERIA
The assay was considered valid if the following criteria were met:
– Blank controls: mean OD value < 0.1.
– Negative controls: mean OD value ≥ 0.6 and ≤ 1.5, SD of % viability ≤ 18.
– Positive controls: mean viability expressed as percentage of the negative control ≤ 40% and SD of % viability ≤ 18.
– Test item: SD of % viability ≤ 18.

Control samples:
other: Positive control: 5% (w/v) sodium dodecyl sulphate (SDS) solution; negative control: Dulbecco’s phosphate buffered saline (D-PBS); Test item without MTT (to evaluate the potential interferring ability of the test item on Optical Density).
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20±2 mg/epidermis unit of solid test item for each replicate (total number of replicates: 3).

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 20 µl/epidermis unit of Dulbecco’s phosphate buffered saline (D-PBS) (total number of replicates: 3).

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 20 µl/epidermis unit of 5%(w/v) sodium dodecyl sulphate (SDS) solution for each replicate (total number of replicates: 3).
- Concentration (if solution): 5 %(w/v) sodium dodecyl sulphate (SDS) solution, obtained by1:1 dilution in sterile water of a sterile commercial 10 %(w/v) SDS solution in water.

NSC living (Test item withou MTT to evaluate the potential colouring interaction of the test item)
- Amount(s) applied (volume or weight with unit): 20 ± 2 mg/epidermis unit of solid test item without MTT for each replicate (total number of replicates: 2).
Duration of treatment / exposure:
15 ± 0.5 minutes in a ventilated cabinet at room temperature. At the end of the exposure, each tissue was rinsed with approximately 25 ml of sterile D-PBS, filling and empting the tissue insert.
Duration of post-treatment incubation (if applicable):
42 ± 1 hour by incubation at 37 °C, 5% CO2 and saturated humidity.
Number of replicates:
3 replicates for negative control, positive control and test item and two replicates for test item without MTT. Note:The test item without MTT is Non Specific Colouring potential (NSC living) using to evaluate the potential colouring of the test item.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean of 3 replicates (A1, A2, A3) for test item (for each replicate the reading of absorbance was performed in duplicate)
Value:
ca. 96
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- RESULTS OF MAIN TEST:
The relative mean viability of the test item treated tissues was 96 % after 15 minutes exposure period and 42 hours of post-exposure incubation period.

- OTHER EFFECTS:
- Direct-MTT reduction: no interation of test item with MTT. Before the main assay a preliminary test was performed to assay the ability of test item to reduce MTT per se. A colourless suspension, with white precipitate, was observed in the MTT solution at the end of the incubation period, indicating that the test item could not interact with MTT.
- Colour interference with MTT: possible colour interference of test item. Before the main assay a preliminary test was performed to assay the ability of test item of colouring the water per se. 20±2 mg of the test item was added to 180 µl of distilled water in at transparent tube and the resulting suspension mixed by using a vortex for 15 minutes. Colouring of the suspension at the end of the incubation time was evaluated by spectral analysis at 595 nm. The value obtained for the Optical Density (OD) was 1.754 (limit OD < 0.1) ,indicating that the test item has a potential interfering ability. Based on the results obtained, an additional control was added in the Main Assay for the evaluation of Non Specific Colouring potential (NSC living). Non Specific Colouring potential (NSC living) is the test item without MTT.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for blank controls: the mean Optical Density of Blank Controls was 0.0, lower than the maximum acceptable value (0.1).
- Acceptance criteria met for negative control: the negative control gave the expected baseline value (Mean of Optical Density values of the three replicates= 0.8 and it is inside the range of acceptability: mean OD value ≥ 0.6 and ≤ 1.5 ) and variability (Standard Deviation for three replicates : 16.2 % lower than the maximum acceptable value 18 %).
- Acceptance criteria met for positive control: the positive control caused the expected cell death (Mean of cell viability of the three replicates = 3% - lower than the maximum acceptable value 40%) and variability (SD of % viability for three replicates equal to 0.5 - lower than the maximum acceptable value 18%).
- Acceptance criteria met for test item :the test item did not induce cell death in any replicate, the mean cell viability after the blank subtraction was 96% when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 12.3 -lower than the maximum acceptable value (18%).
Note: The Non Specific Colour(NSC living) induced by the test item was 0%, thus only the OD-blank background subtraction was performed for the evaluation of irritant properties of Tris(ethyl acetoacetato-O1’,O3)aluminium.

Applicant's summary and conclusion

Interpretation of results:
other: not classified as skin irritant, according to the CLP Regulation (EC n.1272/2008)
Conclusions:
The test item Tris (ethyl acetoacetato-O1’,O3) aluminium is identified as not irritant to the skin according to the CLP Regulation (EC n.1272/2008).
Executive summary:

Tris (ethyl acetoacetato-O1’,O3) aluminium was tested for skin irritation according to OECD TG 439 in vitro Skin irritation Reconstructed Human Epidermis (RhE) MODEL (EPISKIN™/MTT method). The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 ± 1 hour recovery period. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system, being this reaction an index of cell viability.

 

Before the Main Assay, a preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducing MTT per se. A colourless suspension, with white precipitate, was observed in the MTT solution at the end of the incubation period, indicating that the test item could not interact with MTT. In a second step, the test item was assayed for the ability of colouring water per se. A white suspension, without precipitate was observed; spectral analysis of the test item in water, to evaluate the ability of the test chemical to absorb light at 595 nm, was performed. The value obtained for the Optical Density (OD) was 1.754, indicating that the test item has a potential interfering ability (limit value OD < 0.1). Based on these results, an additional control was added in the Main Assay.

 

In the Main Assay, the test item was applied as supplied in three replicates at the treatment level of 20±2 mg/epidermis unit, each measuring 0.38 cm^2 (treatment level: 53 mg/cm^2). Positive and negative controls [a 5% (w/v) sodium dodecyl sulphate solution in water and Dulbecco’s phosphate buffered saline (D-PBS), respectively] were concurrently tested, in the same number of replicates and test conditions at the treatment level of 20 µl/epidermis. In order to verify if the test item results had to be corrected, the Non Specific Colour (NSC living) was evaluated using two alive treated tissues without MTT staining and compared with the D-PBS control.

In the Main Assay, the negative control gave the expected baseline value (Optical Density values of the three replicates higher than 0.6) and variability [Standard Deviation (SD) of % viability lower or equal to 18],in agreement with the guideline indications. According to the method ,the negative control mean value is considered the base line value of the experiment and thus represents 100 % of cell viability. The positive control caused the expected cell death (3 % of cell viability when compared to the negative control) and variability (SD of % viability equal to 0.5). Based on the stated criteria (mean viability ≤ 40% and SD of % viability ≤ 18), the assay was regarded as valid. The Non Specific Colour (NSC living) induced by the test item was 0 %, thus only the OD-blank background subtraction was performed for the evaluation of irritant properties of Tris(ethyl acetoacetato-O1’,O3)aluminium.

 

The test item did not induce cell death in any replicate, the mean cell viability after the blank subtraction was 96 % when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 12.3 (lower than 18, as stated in the Study Protocol).

 

Based on the results obtained, the test item Tris (ethyl acetoacetato-O1’,O3) aluminium is identified as not irritant to the skin according to the UN GHS (for member states that do not adopt optional Category 3) and according to the CLP Regulation (EC n.1272/2008). Note: the criteria for classification regarding skin irritation/corrosion of UN GHS are the same of the CLP Regulation.