Registration Dossier

Administrative data

Endpoint:
skin irritation: 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Jul. 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Commission Regulation (EU) No. 640/2012 amending EU method B.46 -- adopted Jul. 2012
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid: viscous

In vitro test system

Test system:
human skin model
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE:
The test system is a commercially available EpiDermTM-Kit, procured by MatTek. The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell culture inserts.

EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.
Designation of the kit: EPI-200-SIT
Day of delivery: 07. May 2019
Batch no.: 28695


PRE-TESTS:
1) Nylon mesh compatibility:
The test item was tested for possible reaction with the nylon mesh, which is used to ensure sufficient contact with the tissue surface. 30 µL of the test item were pipetted onto a nylon mesh on a microscope slide.
No reaction with the nylon mesh was visible after an exposure time of 1 hour.

2) Assessment of coloured or staining test items:
It was tested whether the test item develops a colour without MTT addition. 30 µL test item were given in a test tube with 0.3 mL H2O demin. and incubated at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity for 1 hour.
The resulting solution was colourless, therefore no binding capacity had to be tested.

3) Assessment of direct reduction of MTT by the test item:
The test item was tested for the ability of direct MTT reduction. To test for this ability, 30 µL test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity for 1 hour. Untreated MTT medium was used as control.
The MTT solution did not change its colour within 1 hour. Therefore, direct MTT reduction by the test item had not taken place and no data correction was necessary.

PRE-INCUBATION OF TISSUES:
All working steps were performed under sterile conditions. For each treatment group (negative control, test item and positive control) a 6-well-plate was prepared with 0.9 mL assay medium in 3 of the 6 wells (upper row). The tissues were inspected for viability. Then, the tissues were transferred into the wells, which contain medium by using sterile forceps and placed into the incubator at 37 ± 1°C and 5 ± 1% CO2 and ≥ 95% relative humidity for 1 hour.
After 1 hour pre-incubation, the other 3 wells of each plate (lower row) were filled with fresh assay medium (0.9 mL). Every tissue was transferred into a well of the lower row. All 6-well-plates were set into the incubator at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity for 19 hours 25 minutes.

TREATMENT:
- One plate (3 tissues) was used as negative control; each tissue was treated with 30 µL DPBS buffer, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
- One plate was used as positive control; each tissue was treated with 30 µL 5% SDS-solution, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
- One plate was used for treatment with the test item: 30 µL test item was applied and a nylon mesh was added in order to ensure sufficient contact with the tissue surface.

Tissues were dosed in 1-minute-intervals. After dosing the last tissue, all plates were transferred into the incubator for 35 minutes at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity. 1 hour after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals. After rinsing thoroughly with DPBS, each tissue was blotted with sterile cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL). The surface of the inserts was then carefully dried with a sterile cotton tipped swab. Then, the tissues were set in the incubator for 23 hours and 32 minutes at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity.

MEDIUM RENEWAL:
After post-incubation, the tissues were removed from the incubator and shaken for 5 minutes (120 rpm). 0.9 mL assay medium were filled in the lower row of the 6-well-plate. Then the inserts were transferred into the lower row of the 6-well-plate and set into the incubator for 18 hours and 25 minutes for post-incubation at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity.

MTT ASSAY:
After a total incubation time of 41 hours and 57 minutes, a 24-well-plate was prepared with 300 µL freshly prepared MTT-solution (1 mg/ml) in each well. The tissues were blotted on the bottom and then transferred into the 24-well-plate. Then the 24-well-plate was set into the incubator for 3 hours and 2 minutes at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity. After this time, the MTT-solution was aspirated and replaced by DPBS buffer. 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 (120 rpm) for 2 hours at room temperature.
After 2 hours, 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, two replicates with 200 µL solution (each) were pipetted into a 96-well-plate which was read in a plate spectrophotometer at 570 nm.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30µL DPBS buffer

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30µL 5% SDS-solution
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
23 hours and 32 minutes
Number of replicates:
2

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of 2 replicates
Value:
124.9
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system:
- Direct-MTT reduction:
- Colour interference with MTT:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The validity of the skin irritation study at LAUS GmbH was demonstrated in a proficiency study. For this purpose, 10 proficiency chemicals (indicated by the OECD 439 guideline) were tested. All of the 10 proficiency chemicals were correctly categorized. Therefore, the proficiency of the skin irritation study was demonstrated.

ACCEPTANCE OF RESULTS:
- Optical density of negative control: 1.5 -- acceptance criterion: ≥ 0.8 and ≤ 2.8 --> accepted
- % Tissue viability of positive control SDS: 3.9% -- acceptance criterion: ≤ 20% of negative control --> accepted
- SD of mean viability of the tissue replicates (%): 9.5% (negative control), 0.8% (positive control), 14.3 (test item) -- acceptance criterion ≤ 18% --> accepted

Any other information on results incl. tables

Historical data

 Parameter

Negative Control

Optical Density

 Positive control

% OD compared to negative control

 

DPBS buffer

5% SDS solution

Mean 

1.788 

 4.4%
Standard deviation  0.320 3.0% 
 Range 0.476 - 2.471   1.7 - 17.1%
 This study 1.549  3.9% 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item 'Oligomerisation product obtained by plasma treatment of vegetable oil mono- and polyunsaturated C16-C22 triglycerides' is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.
Executive summary:

Three tissues of the human skin model EpiDermTM were treated withthe test itemfor 60 minutes. The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2; as indicated by the supplier). DPBS-buffer was used as negative control and 5% SDS solution was used as positive control.

After treatment with the negative control, the mean absorbance value was within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.5. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 3.9% (required: ≤

20%). The variation within the tissue replicates of negative control, positive control and test item was acceptable (required: ≤ 18%).

 

After the treatment with the test item, the mean value of relative tissue viability was increased to 124.9 %. This value is above the threshold for skin irritation potential (50%). Test items that induce values above the threshold of 50% are considered non-irritant to skin.

 

Therefore, the test item 'Oligomerisation product obtained by plasma treatment of vegetable oil mono- and polyunsaturated C16-C22 triglycerides' is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.