[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 October 2017 to 26 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU-Method B.40 BIS. “IN VITRO SKIN CORROSION: HUMAN SKIN MODEL TEST“

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Octadecanoic acid, reaction products with tetraethylenepentamine

Cas Number:

71799-54-7

Molecular formula:

Not applicable - UVCB substance

IUPAC Name:

Octadecanoic acid, reaction products with tetraethylenepentamine

Test material form:

solid

Details on test material:

- Appearance: Amber solid
- Storage: Room temperature (20 ± 5 °C); protected from light.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended test system in international guidelines.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm-Kit
- Source: MatTek In Vitro Life Science Laboratories, Bratislava.
- Tissue batch number(s): 25848 (main test) and 25835 (additional test).
- Delivery date: 10. Oct. 2017 (main test) and 08. Aug. 2017 (additional test).

PRE-TESTS
- Assessment of Coloured or Staining Test Materials: It was tested whether the test material develops a colour without MTT addition. 25.0 mg of the test material were given in a test tube with 0.3 mL demineralised water and incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 for 1 hour. The resulting solution was colourless, therefore no binding capacity had to be tested.
- Assessment of Direct Reduction of MTT by the Test Material: The test material was also tested for the ability of direct MTT reduction. To test for this ability, 26.1 mg of the test material were added to 1 mL of MTT solution and the mixture was incubated in the dark at 37 ± 1 °C and 5.0 ± 0.5 % CO2 for 1 hour. Untreated MTT solution was used as control. The MTT solution changed its colour to purple within 1 hour.
- Additional Test: Direct Reduction of MTT with freeze killed Tissues: As the test material had shown its ability to reduce MTT in the pre-test, it was necessary to perform a functional test with freeze killed tissues that possess no metabolic activity but absorb and bind the test material like viable tissues. Freeze killed tissues were prepared by placing untreated tissues in freezer (– 20 ± 5 °C) over night. Once killed, the tissues were stored indefinitely in the freezer. In addition to the normal test procedure described, the functional check employed two freeze-killed tissues treated with the MTT reducing test material (for 3 minutes and 1 hour experiment) and two untreated killed tissues (for 3 minutes and 1 hour experiment) that showed the small amount of MTT reduction due to residual NADH and associated enzymes within the killed tissues. Therefore, direct MTT reduction had taken place and data correction was necessary.

PREPARATIONS
- On the day of the start of the experiment, the MTT concentrate was thawed. The concentrate was diluted with the assay medium directly before use.
- The tissue plate was brought out of the fridge 1 hour before the treatment.
- The assay medium was warmed in the water bath to 37 ± 1 °C.

MAIN TEST
- Four 6-well-plates were prepared with 0.9 mL assay medium in each well. The inserts containing the tissues were transferred to the wells using sterile forceps and the 6-well-plates were set into the incubator at 37 ± 1 °C and 5.0 ± 0.5 % CO2 for 1 hour (pre-incubation).
- For each experiment (“3 minutes” and “1 hour”), one 24-well-plate was prepared as holding plate. 12 wells of each plate were filled with 300 μL assay medium, the other 12 with 300 μL MTT solution. One additional plate was left empty. The plates were stored in the incubator at 37 ± 1 °C and 5.0 ± 0.5 % CO2.
- For each experiment (“3 minutes” and “1 hour”), two 6-well-plates for the assay were used. After pre-incubation, the assay medium was replaced by fresh assay medium and the test was started, using two wells as negative control with 50 μL demineralised water, two wells as positive controls with 50 μL potassium hydroxide solution and two other wells for testing the test material.
- Defined amounts of the test material were applied together with 25 μL demineralised water. Main Test: 26.0 and 26.2 mg (3 minute incubation), 25.9 and 25.5 mg (1 hour incubation). Additional test: 26.9 and 27.1 mg (3 minute incubation), 26.9 and 25.3 mg (1 hour incubation).
- At the start of each experiment (application of negative controls), a stop watch was started.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C
- Temperature of post-treatment incubation: 37 ± 1 °C (with MTT)

NUMBER OF REPLICATE TISSUES: 2

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the respective incubation time (“3 minutes” and “1 hour”) at 37 ± 1 °C and 5.0 ± 0.5 % CO2, the inserts were removed from the plates using sterile forceps. The inserts were thoroughly rinsed with DPBS, blotted with sterile cellulose tissue and set into the respective holding plate, using the wells containing assay medium.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- After transfer of all inserts, they were immediately moved to the wells containing MTT medium, blotting the bottom with cellulose tissue again before setting the insert into the MTT well. The tissues were incubated with MTT solution for 3 hours at 37 ± 1 °C and 5.0 ± 0.5 % CO2.
- After this time, the MTT solution was aspirated and replaced by DPBS. This was then aspirated, too, and replaced several times. At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was then shaken for 2 hours at room temperature.
- Afterwards, the inserts were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation.
- From each well, three replicates with 200 μL solution (each) were pipetted into a 96-wellplate which was read in a plate spectrophotometer at 570 nm.

EVALUATION
The photometric absorbance of the negative controls was considered as 100 %. For the mean of the 3 replicates of test item and positive control, tissue viability was calculated as % photometric absorbance compared to the negative control.

CALCULATIONS
Calculations were performed as follows:
- Calculation of mean OD of the blank isopropanol (ODBlk)
- Subtraction of mean ODBlk of each value of the same experiment (corrected values)
- Calculation of mean OD of the two replicates for each tissue
- Calculation of mean OD of the two relating tissues for controls and test material
(Corrected OD value of negative control corresponds to 100 % viability)

% Viability = [ODcorrected if the test material or positive control/ OD corrected of mean negative control]·100

- Calculation for Additional Test (Correction)
OD Test Material (corrected) = OD Test Material (Main Test) - (mean OD test material with freezekilled tissues – mean OD negative control with freeze-killed tissues)

PREDICTION MODEL / DECISION CRITERIA
- Corrosivity was assessed using the following:
STEP 1
% tissue viability after 3 min. incubation time < 50 % of negative control = corrosive to skin
% tissue viability after 3 min. incubation time ≥ 50 % of negative control and % tissue viability after 1 h incubation time < 15 % of negative control = corrosive to skin
% tissue viability after 3 min. incubation time ≥ 50 % of negative control and % tissue viability after 1 h incubation time ≥ 15 % of negative control = non-corrosive to skin

STEP 2 (for substances/mixtures identified as corrosive in STEP 1)
% tissue viability after 3 min. incubation time < 25 % of negative control = GHS Skin Corrosive Sub-category 1A
% tissue viability after 3 min. incubation time ≥ 25 % of negative control = GHS Skin Corrosive Sub-categories 1B or 1C.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25.3 - 27.1 mg

NEGATIVE CONTROL
- Amount(s) applied: 50 μL

POSITIVE CONTROL
- Amount(s) applied: 50 μL
- Concentration: 8 M

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

3 hours with MTT

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

106.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure

Value:

101.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

MEASURED VALUES OF THE MAIN TEST
- As blank, the optical density of isopropanol was measured in 12 wells of the 96-well-plate, this gave a mean of 0.039. The mean absorbance of isopropanol was subtracted from the values of the test material, positive control and negative control. The corrected mean and relative standard deviation (RSD) of the tissue replicates were also calculated. Results can be seen in Table 1.

MEASURED VALUES OF THE ADDITIONAL TEST
- As blank, the optical density of isopropanol was measured in 12 wells of the 96-well-plate, this gave a mean of 0.037. The mean absorbance of isopropanol was subtracted from the values from the measured absorbances of the freeze-killed tissues. The corrected mean and relative standard deviation (RSD) of the freeze-killed tissue replicates were also calculated. Results can be seen in Table 2.
- The mean value of the test material (freeze-killed tissues) was subtracted from the mean value of the negative control (freeze-killed tissues). This difference was -0.009 (3 minutes) and 0.019 (1 hour). These values were subtracted from the absorbance value of the test material in the main test to achieve the corrected absorbance values.
- Corrected values can be seen in Table 3.

CORROSIVITY OF THE TEST MATERIAL
- The mean value of relative tissue viability of the test material was increased to 106.9 % after 3 minutes treatment. This value is above the threshold for corrosivity (50 %).
- After 1 hour treatment, the mean value of relative tissue viability of the test material was increased to 101.4 %, lying above the threshold for corrosivity (15 %). Therefore, the test material is considered as not corrosive to skin.

VALIDITY OF THE TEST
- The criterion for optical density of the negative control (≥ 0.8 and ≤ 2.8) was fulfilled: optical density was 1.9 (3 minutes) and 1.8 (1 hour). The positive control showed clear corrosive effects. The criterion for the viability of the 1 hour experiment, expressed as % of the negative control (< 15 %), was fulfilled, too. The mean value of relative tissue viability was 7.4 %.
- Values for negative control and for positive control were within the range of historical data of the test facility. Therefore the experiment is considered valid.

Any other information on results incl. tables

Table 1: Mean Absorbance Values of the Main Test

Designation	Exposure time
	3 minutes			1 hour
	Negative Control	Test Material	Positive Control	Negative Control	Test Material	Positive Control
Mean – blank (tissue 1)	1.888	1.999	0.411	1.822	1.621	0.129
Mean – blank (tissue 2)	1.921	2.056	0.442	1.709	1.996	0.134
Mean of the two tissues	1.905	2.027	0.426	1.765	1.808	0.131
RSD	1.2 %	2.0 %	5.1 %	4.5 %	14.7 %	2.9 %

 

Table 2: Mean Absorbance Values of the Additional Test

Designation	Exposure time
	3 minutes		1 hour
	Negative Control	Test Material	Negative Control	Test Material
Mean – blank (tissue 1)	0.090	0.077	0.072	0.085
Mean – blank (tissue 2)	0.087	0.084	0.068	0.094
Mean of the two tissues	0.089	0.080	0.070	0.089
RSD	1.9 %	6.2 %	4.4 %	7.1 %

 

Table 3: Mean Corrected Absorbance Values of the Experiment

Designation	Test Material Exposure Time
	3 minutes	1 hour
Mean – blank (tissue 1)	2.008	1.602
Mean – blank (tissue 2)	2.065	1.977
Mean of the two tissues	2.036	1.789
RSD	2.0 %	14.8 %

Table 4: Percentage Tissue Viabilities (%)

Exposure Time	Test Material	Positive Control
3 Minutes	106.9	22.4
1 Hour	101.4	7.4

Applicant's summary and conclusion

Interpretation of results:

other: Not corrosive in accordance with EU criteria

Conclusions:

Under the conditions of this study, the test material is considered to be non-corrosive to the skin.

Executive summary:

The potential of the test material to cause corrosion to the skin was determined in vitro, in accordance with the standardised guidelines OECD 431 and EU Method B 40bis, under GLP conditions using the Reconstructed Human Epidermis (RHE) Test Method.

One valid experiment was performed. In the pre-test the test material showed MTT-reducing properties. The probability to influence the photometric measurement had to be excluded. Therefore an additional test using freeze-killed tissues that possess no metabolic activity but absorb and bind the test material like viable tissues was performed. The result of the additional test showed that MTT reduction by the test material did not influence the result of the study.

In the main test two tissues of the human skin model EpiDerm were treated with the test material for 3 minutes and 1 hour, respectively. Demineralised water was used as negative control and 8 M KOH was used as positive control.

After treatment, the respective substance was rinsed from the tissues. Then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to a blue formazan. Formazan production was evaluated by measuring the optical density (OD) of the resulting formazan solution.

After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals thus showing the quality of the tissues. The OD was 1.9 (3 minutes experiment) and 1.8 (1 hour experiment).

The positive control showed clear corrosive effects for both treatment intervals. The mean relative tissue viability value was reduced to 7.4 % for the 1 hour treatment. After 3 minutes treatment with the test material, the mean value of relative tissue viability was increased to 106.9 %. This value is above the threshold for corrosion potential (50 %). After 1 hour treatment, mean value of relative tissue viability was increased to 101.4 %. This value, too, is above the threshold for corrosion potential (15 %).

Under the conditions of the study, the test material was considered to be non-corrosive to the skin.