Octadecanoic acid, reaction products with triethylenetetramine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-05-15 to 2017-05-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015-07-28

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Octadecanoic acid, reaction products with triethylenetetramine

EC Number:

270-171-6

EC Name:

Octadecanoic acid, reaction products with triethylenetetramine

Cas Number:

68412-15-7

Molecular formula:

C24H54N4O2

IUPAC Name:

N'-[2-(2-aminoethylamino)ethyl]ethane-1,2-diamine;octadecanoic acid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

recommended by the OECD testing guideline 439

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SYSTEM
EPISKIN™ - 0.38 cm2, Supplier SkinEthic Laboratories (4, A. Fleming, 69366 Lyon, France), Batch 17-EKIN-020, arrived at RTC on 16 May 2017.
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.
The test system was shipped on Monday and received on Tuesday. According to the supplier procedure, at arrival, 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.

REMOVAL OF TEST SUBSTANCE
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.

PRELIMINARY TEST FOR DIRECT MTT REDUCTION
Non-specific reduction of MTT was evaluated as follows: 2 mL of MTT ready-to-use solution (0.3 mg/mL) was incubated with 20 ± 2mg 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.

PRELIMINARY TEST FOR COLOURING POTENTIAL
The test item's colouring potential was assessed for potential interaction with the test system. 20 ± 2mg of the test item was added to 180 µL of distilled water in a transparent tube and the resulting solution/suspension mixed by using a vortex for 15 minutes. Suspension appearance was recorded.

MTT STAINING FOR MAIN ASSAY
Each tissue insert was incubated with 2mL/well of MTT ready-to-use solution. Plates were incubated for approximately 3 hours at 37 °C, 5% CO2 and saturated humidity, simulating test conditions. 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.
Observation of blue or purple appearance of the solution at the end of the incubation time was carried out as follows: 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 (13000 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 (200 µL) of acidic isopropanol were analysed and used as blank. In order to ensure the spectrophotometer linear range, on the day of spectrophotometer analysis, an MTT formazan calibration curve was performed and adequate results were obtained.

PREDICTION MODEL / DECISION CRITERIA
A test substance is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test substance is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST ITEM: 20 ± 2 mg/epidermis unit, each one measuring 0.38cm2 (treatment level: 53mg/cm2)
NEGATIVE CONTROL: 20 µL/epidermis unit
POSITIVE CONTROL: 20 µL/epidermis unit

Duration of treatment / exposure:

15 min (at room temperature)

Duration of post-treatment incubation (if applicable):

A 42 ± 1 hour recovery period was allowed by incubation at 37°C, 5% CO2 and saturated humidity.

Number of replicates:

triplicates

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

test item

Value:

97

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:

- OTHER EFFECTS:
In a first pre-test, the test item was assayed for the ability of reducing MTT per se. A pale grey precipitate was observed at the end of the incubation period when the test item was added to the MTT solution, indicating that the test item could directly interact with MTT.

In a second pre-test, the test item was assayed for the ability of colouring water per se. A white suspension was observed and the Optical Density (OD) measured at 595 nm was 0.967, indicating that the test item has a potential interfering ability.

Based on these results, additional controls were added in theMain Assay.

In order to verify if the test item results had to be corrected, the non specific colour (NSC) was evaluated using two alive treated tissues without MTT staining and compared with the D-PBS control. Moreover, non specific MTT reduction (NSMTT) was evaluated using two killed tissues and compared with negative control performed with alive tissues. Since the test item is able both to stain tissues and reduce MTT, to avoid a possible double correction for colour interference, a third control for Non Specific Colour in killed tissues (NSCkilled) was performed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for blank: yes
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

The results of the main assay are:

Group	OD 595 nm Tissue 1	OD 595 nm Tissue2	OD 595 nm Tissue3	Mean OD allTissues	Standard Deviation	Viability [%]
Negative Control	0.6245	0.6505	0.6240	0.6	0.02	100
Positive Control	0.0410	0.0295	0.0465	0.04	0.01	6
Test item	0.5580	0.5570	0.7190	0.6	0.09	97

The meanOpticalDensity of Blank Controls was 0.037, lower than the maximum acceptable value (0.1).

The negative control gave the expected baseline value (mean Optical Density value ≥0.6 and ≤1.5) and variability (Standard Deviation of the % viability lower or equal to 18), in agreement with guideline indications.

According to the method, the mean value is considered the baseline value of the experiment and thus represents 100% of cell viability.

Positive control results indicated an appropriate cell death with an acceptable relative cell viability (6% of the negative control value).

Variability between replicates gave also the expected value (SD of%viability = 1.4).

Based on the stated criteria, mean viability, expressed as percentage of the negative control, lower or equal to 40% and standard deviation of % viability equal or lower than 18, the study was accepted as valid.

The colouring interference (NSC) was 1%, as well as the non specific MTT reduction (NSMTT), thus only the blank subtraction was performed.

The test item did not induce cell death in any replicate, the mean cell viability after the appropriate subtractions was 97% when compared to the negative control.

Intra-replicate variability was acceptable with a SD of % viability value equal to 14.7 (lower than 18, as stated in the Study Protocol).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Octadecanoic Acid, reaction products with triethylenetetramine was not irritating in the in vitro skin irritation test.

Executive summary:

The potential of the test item Octadecanoic acid, reaction products with triethylenetetramine to be irritant to the skin was investigated according to OECD Guideline 439 (28 July 2015) through an in vitro skin irritation study, using a commercial reconstructed human epidermis (RhE) model named EPISKIN™.

The test item Octadecanoic acid, reaction products with triethylenetetramine (100% a.i.) was tested for its ability to impair cell viability. The test item was applied as supplied by the sponsor in three tissue replicates at the treatment level of 20 ± 2 mg/epidermis unit, each one measuring 0.38cm²(treatment level: 53mg/cm²).

 

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 pale gray precipitate was observed at the end of the incubation period when the test item was added to the MTT solution, indicating that the test item could directly interact with MTT. In a second step, the test item was assayed for the ability of colouring water per se. A white suspension was observed and the Optical Density (OD) measured at 595 nm was 0.967, indicating that the test item has a potential interfering ability. Based on these results, additional controls were added in the Main Assay

 

After an exposure period of 15 minutes at room temperature, each tissue was rinsed with approximately 25 mL of sterile D-PBS to remove any residual test material. Subsequently, the tissues were incubated for 42 h at 37 °C. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive controls [sodium dodecyl sulphate 5% (w/v) solution in water] and the negative controls [Dulbecco’s phosphate buffered saline (D-PBS)] were concurrently tested, in the same number of replicates and test conditions as the test item. The controls were tested at the treatment level of 20µL/epidermis unit and gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

 

In order to verify if the test item results had to be corrected, the non specific colour (NSC) was evaluated using two alive treated tissues without MTT staining and compared with the D-PBS control. Moreover, non specific MTT reduction (NSMTT) was evaluated using two killed tissues and compared with negative control performed with alive tissues. Since the test item is able both to stain tissues and reduce MTT, to avoid a possible double correction for colour interference, a third control for Non Specific Colour in killed tissues (NSCkilled) was performed.

 

The colouring interference (NSC) was 1%, as well as the non specific MTT reduction (NSMTT); thus only the black subtraction was performed.

The test item did not induce cell death in any replicate. When compared to the negative control, the mean cell viability of the tissues treated with the test item was 97% after the appropriate subtractions.

Based on the results obtained, and since the mean cell viability of the treated tissues was above 50%, the test item Octadecanoic acid, reaction products with triethylenetetramine is classified as not irritant to the skin (UN GHS No Category).