Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 07, 2017 to August 11, 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Batch No.: WD0066442; Purity: 100 %; Appearance: homogeneous amber liquid

In vitro test system

Test system:
human skin model
Remarks:
EpiDermTM tissues
Source species:
other: human-derived epidermal keratinocytes
Cell type:
non-transformed keratinocytes
Cell source:
other:
Vehicle:
unchanged (no vehicle)
Details on test system:
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.

Origin:
EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.
Designation of the kit: EPI-200-SIT
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
One plate 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 substance:
For each plate, 3 tissues were used. The tissues were wetted with 25 μL DPBS buffer before applying the test substance and spreading it to match the tissue size.
The following amounts were applied to the tissues:
Replicate 1: 26.6 mg
Replicate 2: 26.2 mg
Replicate 3: 26.7 mg

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 ± 0.5% CO2. 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, each tissue was blotted with sterile cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL). Then, the tissues were set in the incubator for 23 hours 30 minutes at 37 ± 1°C and 5.0 ± 0.5% CO2.

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-wellplate. Then the inserts were transferred into the lower row of the 6-well-plate and set into the incubator for 19 hours 5 minutes for post-incubation at 37 ± 1 °C and 5.0 ± 0.5% CO2.
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
23 hours and 30 minutes
Number of replicates:
3 replicates

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
72.4
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Results:
% Tissue viability (tissue 1): 71.5 %
% Tissue viability (tissue 2): 73.1 %
% Tissue viability (tissue 3): 72.6 %
% Tissue viability (mean) 72.4 %

Tissue Viability

The photometric absorbance of the negative controls is considered as 100%. For each replicate of test substance and positive control, tissue viability is calculated as % photometric absorbance compared with the mean of the negative controls:
% Tissue Viability = (OD replicate test substance resp. positive control x 100%) / OD negative controls

Acceptance of the results:

- The test substance is considered as non-irritant to skin.
- After the treatment, the relative absorbance values were reduced to 99.4%. This value is above the threshold for skin irritation (50%).
- The optical density of the negative control was well within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8.
- The positive control has met the acceptance criterion too, demonstrating the validity of the test system.
- Variation within replicates was within the accepted range for negative control, positive control and test substance (required: ≤ 18%).

Validity:
- OD of negative control: 1.6 (Required: ≥ 0.8 and ≤ 2.8)
- % Tissue viability of positive control SDS: 2.2% (Required: ≤ 20 % of negative control)
- SD of mean viability of the tissue replicates (≤18 %):

9.7% (negative control)
0.2% (positive control)
0.8% (test substance)

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
non-irritant
Conclusions:
Under the study conditions, after the treatment with the test substance, the mean value of relative tissue viability was reduced to 72.4%. This value is above the threshold for skin irritation potential (50%). Therefore, the test substance is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) test method.
Executive summary:

A study was conducted to determine the skin irritation potential of the test substance using the Reconstructed Human Epidermis (RHE) Test Method according to OECD Guideline 439 and EU Method B.46, in compliance with GLP. One valid experiment was performed. Three tissues of the human skin model EpiDermTM were treated with the test substance for 60 minutes. The test substance was applied directly to each tissue and spread to match the tissue size (0.63 cm2). DPBS-buffer was used as negative control and 5% SDS solution was used as positive control. After treatment with the negative control, the absorbance values with a mean OD of 1.6, were within the required acceptability criterion of 0.8≤ mean OD ≤2.8. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 2.2%. The variation within the tissue replicates of negative control, positive control and test substance was acceptable (required: ≤ 18%). Under the study conditions, after the treatment with the test substance, the mean value of relative tissue viability was reduced to 72.4%. This value is above the threshold for skin irritation potential (50%). Therefore, the test substance is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) test method (Andres, 2017).