Registration Dossier

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:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
July 2015
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 EpiDermTM 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.