Aluminium triethanolate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Aluminium triethanolate

EC Number:

209-105-8

EC Name:

Aluminium triethanolate

Cas Number:

555-75-9

Molecular formula:

C2H6O.1/3Al

IUPAC Name:

aluminum triethanolate

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Purchased or derived from tissue obtained from accredited institutions.

Source strain:

not specified

Details on animal used as source of test system:

SOURCE ANIMAL
- Source: Reconstructed human epidermis tissue containing normal human keratinocytes. In all cases, consent was obtained by the institutions from the donor for use of the tissues for research purposes. The cells used to create the tissues were screened for potential biological contaminants. Tissue function and barrier function tests were also conducted indicating the tissues suitability.

Justification for test system used:

Standard as per OECD guideline

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM Tissues (0.63cm2) - strain 00267
- Tissue batch number(s): 28690
- Date of initiation of testing: 03 April 2019

Test for direct MTT reduction:
As specified, a test item may interfere with the MTT endpoint, if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test item was checked for the ability to directly reduce MTT according to the procedure below: 25 mg of the test item was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control. If the MTT solution containing the test item turned blue/purple relative to the control, the test item was presumed to have reduced the MTT. The test item was shown to directly reduce MTT in the direct MTT reduction test. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin corrosion potential was performed in parallel on viable and non-viable, freeze-killed, tissues. This step was a functional check which employs freeze-killed tissues that possess no metabolic activity but absorb and bind the test item like viable tissues. Freeze-killed tissues were prepared prior to the study by placing untreated EPIDERMTM tissues in an empty 12-well plate and storing in a freezer (-14 to -30 °C) for a minimum of 24 hours. Before use each tissue was thawed by placing in 0.9 mL of assay medium for approximately 1 hour at room temperature. In addition to the normal test procedure, the MTT reducing test item was applied to two freeze-killed tissues per exposure period. In addition, two freeze-killed tissues per exposure period remained untreated. The untreated freeze-killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissues.

Assessment of colour interference:
A test item may interfere with the MTT endpoint if it is colored or if it becomes colored when in wet or aqueous conditions. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed. 25 mg of test item was added to 300 µL of sterile water. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. A visual assessment of the color was then made.

Main test preincbation:
The assay medium was brought to room temperature before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3-Minute and 60-Minute exposure periods. EpiDerm™ tissues were transferred into the 6-well plates containing the assay medium. The 6-well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5% CO2) for approximately 1 hour before dosing.

Application of test item and rinsing: Before pre-incubation was complete, a 24-well plate was prepared for use as a “holding plate” for both the 3-Minute and 60-Minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT-loading. Another 24-well plate was prepared for the MTT-loading. 300 µL of either pre-warmed assay medium (holding plate) or MTT medium (MTT-loading plate) was dispensed into each well. The two plates were placed into the incubator until required. After pre-incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3-Minute exposure period was returned to the incubator, while the other was being dosed for the 60-Minute exposure. For the 60-Minute exposure period, 50 µL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 25 mg of the test item and 50 µL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. 25 µL of sterile water was added for wetting of the test item to increase tissue surface contact. The plate was returned to the incubator (37 °C, 5% CO2) for the 60-Minute exposure period. When dosing for the 60-Minute exposure period was complete, the same procedure was repeated for the 3-Minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24-well plate prepared for MTT-loading. The plate was incubated (37 °C, 5% CO2) for 3 hours. Once the 60-Minute exposure period was complete, the same rinsing and MTT-loading procedure was repeated.
After the 3-Hour MTT incubation was complete, the tissue inserts were blotted and transferred to 24-well plates for formazan (reduced MTT) extraction. The formazan was extracted from the top and bottom of the tissue by completely immersing the tissue insert in 2 mL of isopropanol. The plate was covered with plate sealer, to prevent isopropanol evaporation, and stood overnight at room temperature, to allow extraction to proceed.

Absorbance/optical density measurements: After extraction, each tissue was pierced with a pipette fitted with a 1000 µL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 µL aliquots of the extract were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 µL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 570 nm (OD570) of each well was measured using the Labtech LT-4500 microplate reader and LT-com analysis software.

Evaluation: The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3 and 60-Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water. The relative mean viabilities were calculated in the following way: Relative mean viability (%) = (mean OD570 of test item / mean OD570 of negative control) x 100

Quality criteria: Negative control: The absolute OD570 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD570 of the two negative control tissues should be = 0.8 and = 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.

Positive control: Potassium Hydroxide 8.0N solution is used as a positive control. An assay meets the acceptance criterion if mean relative tissue viability of the 60-Minute positive control is < 15%.

Coefficient of variation: In the range 20 and 100% viability, the Coefficient of Variation between tissue replicates should be = 30%.

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL sterile distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL of 8.0 N Potassium hydroxide

Duration of treatment / exposure:

3 minute and 60 minute exposure periods

Duration of post-treatment incubation (if applicable):

Not applicable

Number of replicates:

Prepared in duplicate

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

86.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure

Value:

91.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Direct MTT reduction: The MTT solution containing the test item turned purple. Therefore, an assessment found the test item was able to directly reduce MTT and an additional procedure using freeze-killed tissues was performed. However, the results obtained showed that no interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results or for reporting purposes.

Assessment of colour interference: The solution containing the test item was a white color. This color was attributed to the intrinsic color of the test item itself. It was therefore unnecessary to run color correction tissues.

Quality criteria: The mean OD570 for the negative control treated tissues was 1.786 for the 3-Minute exposure period and 1.837 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied. The relative mean tissue viability for the positive control treated tissues was 4.1% relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied. In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Mean OD570 values and viabilities for the negative control item, positive control item and test item:

Tissue	Exposure period	Mean OD570 of individual tissues	Mean OD570 of duplicate tissues	Standard deviation	Coefficient of variation (%)	Relative mean viability (%)
Negative control	3 minutes	1.860	1.786	0.105	5.9	100
		1.712
	60 minutes	1.816	1.837	0.029	1.6
		1.857
Positive control	3 minutes	0.095	0.083	0.018	Na	4.6
		0.070
	60 minutes	0.084	0.075	0.013	Na	4.1
		0.066
Test item	3 minutes	1.600	1.538	0.088	5.7	86.1
		1.475
	60 minutes	1.661	1.689	0.039	2.3	91.9
		1.716

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results obtained, the test item was considered non-corrosive to skin.