Octadecanoic acid, reaction products with acetic acid and tetraethylenepentamine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-04-13 to 2018-04-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

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:

July 2015

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Octadecanoic acid, reaction products with acetic acid and tetraethylenepentamine

EC Number:

271-547-2

EC Name:

Octadecanoic acid, reaction products with acetic acid and tetraethylenepentamine

Cas Number:

68585-02-4

Molecular formula:

C28H63N5O4

IUPAC Name:

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

Test material form:

solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm
- Tissue batch number(s): 25899
- Certificate date: 2018-04-25


TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: The incubation conditions were 37°C, 5% CO2 and 95% relative humidity for the first 35 minutes followed by 25 minutes at room temperature under a sterile hood.
- Temperature of post-treatment incubation (if applicable): 42 hours

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: no details

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Tecan Sunrise Magellan Version 7.2
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: MTT WC assay (OD 540 -570 nm) passed
- Barrier function: ET50 assy (ET50 4.77 -8.72 hrs) passed
- Morphology: passed
- Contamination: sterility an d biological contaminats tested: passed
- Reproducibility: validity criteria fullfilled

NUMBER OF REPLICATE TISSUES: 3 tissues

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Not applicable, no possible interference with the MTT measurement

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: one

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
According to the EU and GHS classification (H314 or H315 / Category 1/2 or no label), an irritant is predicted if the mean relative tissue viability of three individual tissues exposed to the test substance is reduced below or equal to 50% of the mean viability of the negative controls.

mean tissue viability ≤ 50% Irritant (I), (H314 or H315 or GHS Category 1 or 2 )
mean tissue viability > 50% non-irritant (NI).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
25 mg of test item were applied to the skin model.
The test item was grounded and applied as a fine powder.
For better contact of the test item to the skin, the skin surface was moistened with 25 µL Dulbecco’s phosphate buffered saline (D-PBS)

NEGATIVE CONTROL
D-PBS was used as the negative control.
30 µL

POSITIVE CONTROL
5% aqueous sodium dodecyl sulphate (SDS)
30 µL

Duration of treatment / exposure:

The whole exposure period for the used EpiDermTM skin model was 60 minutes. The incubation conditions were 37°C, 5% CO2 and 95% relative humidity for the first 35 minutes followed by 25 minutes at room temperature under a sterile hood.

Duration of post-treatment incubation (if applicable):

Post treatment incubation period of the rinsed tissues in fresh assay medium of 42 hours

Number of replicates:

3 tissues

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test substance

Value:

79.5

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:

The mean viability of cells exposed to the test item was 79.5% 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%.
The test substance was considered to be non-cytotoxic and predicted to be non-irritant to skin.

The mean optical density (OD) of 3 negative control tissues was 1.747 and was well within the acceptable range of ≥ 0.8 to ≤ 2.8.
The viability of cells treated with the positive reference item, 5% SDS, was 6.6% of the negative control and fulfilled the acceptance criterion of ≤ 20%.
The standard deviation (15.7%) determined for all triplicates w3as below the limit of acceptance of 18%.
Hence, all acceptance criteria were met.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the present test conditions, Octadecanoic acid, reaction products with acetic acid and tetraethylenepentamine tested at an exposure time of 60 minutes and a 42-hour post-treatment incubation period, was non-cytotoxic and, hence, predicted to be non-irritant to skin in an experiment employing an artificial three-dimensional model of human skin (EpiDerm™). Hence, the test item did not show irritant properties and is therefore not classified as irritant (UN GHS no category)..

Executive summary:

The EpiDerm^TM model was used to distinguishes irritants in accordance with UN GHS Category 1 or Category 2 from non-classified test substances.

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.

Octadecanoic acid, reaction products with acetic acid and tetraethylenepentamine was applied as solid test item to the model skin surface, which was moistened with Dulbecco’s phosphate buffered saline (D-PBS). D-PBS was used as the negative control. 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item. An exposure time of 60 minutes was employed followed by a 42-hour post-treatment incubation period in fresh medium.

The mean viability of cells exposed to the test substance was 79.5% 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%.Octadecanoic acid, reaction products with acetic acid and tetraethylenepentaminewas considered to be non-cytotoxic and predicted to be non-irritant to skin.

The mean optical density (OD) of 3 negative control tissues was 1.747 and was well within the acceptable range of ≥ 0.8 to ≤ 2.8.

The viability of cells treated with the positive reference item, 5% SDS, was 6.6% of the negative control and fulfilled the acceptance criterion of ≤ 20%.

The standard deviation (15.7%) determined for all triplicates was below the limit of acceptance of 18%.

Hence, all acceptance criteria were met.