Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 February 2018 - 01 March 2018
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:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

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

In vitro test system

Test system:
human skin model
Remarks:
EpiSkinTM
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified
Source strain:
not specified
Justification for test system used:
The assessment of skin irritation has typically involved the use of laboratory animals (OECD TG 404). In relation to animal welfare concerns, TG 404 recommends the use of a tiered testing strategy for the determination of skin corrosion and irritation which includes the use of validated in vitro or ex vivo test methods avoiding pain and suffering of animals.
One of the validated in vitro test methods adopted by the OECD TG 439 makes use of reconstructed human epidermis (RhE) which closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM kit
- Tissue batch number(s): 18-EKIN-009
- Delivery date: 27 February 2018
- Expiration date: 05 March 2018
- Date of initiation of testing: 23 February 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: PBS, volumen no specified
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours at 37°C, 5% CO2
- Spectrophotometer: TECAN Infinite® M200 Microplate Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Historical negative control mean OD range = 0.58630-1.13620 (15 min exposure).
- Histology scoring: 21.5 ± 1.2 (CV = 5.7%), specification ≥ 19.5.
- Barrier function: IC50 = 2.1 mg/mL (specification, 1.5 mg/mL ≤ IC50 ≤ 3 mg/mL)
- Morphology: Well-differenciated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
- Contamination: no

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: no interference.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the tissue viability after after exposure and post-treatment incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the tissue viability after exposure and post-treatment incubation is greater than 50%.


Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 μL (26 μL/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5% SDS
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
100.19
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Remarks:
(PBS)
Positive controls valid:
yes
Remarks:
6.87% viability (5% SDS)
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes. A demonstration of proficiency was performed for the EpiSkinTM kit. Adequate results were obtained for the evaluated chemicals.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, OD mean is 0.69413 (acceptability criteria, 0.6≤OD≤1.5). SD of the negative control group was 0.00378 (acceptablility criteria, SD ≤ 18%).
- Acceptance criteria met for positive control: yes, SD of the positive control group was 0.21% (acceptablility criteria, SD ≤ 18%)
- Acceptance criteria met for variability between replicate measurements: yes. SD of test item was 1.23% (acceptablility criteria, SD ≤ 18%).

Any other information on results incl. tables

Table 1. Mean OD Values of Individual Epidermis Units

 

Absorption (OD570)

 

R1

R2

R3

Mean

Negative control

0.73520

0.73360

0.74080

 

0.73653

Positive control

0.09110

0.09080

0.08845

 

0.09012

Test Item

0.74580

0.73885

0.72885

 

0.73783

Table 2. True OD Values of Individual Epidermis Units

 

Absorption (OD570)

R1

R2

R3

Mean

SD

Negative control

0.69280

0.69120

0.69840

0.69413

0.00378

Positive control

0.04870

0.04840

0.04605

0.04772

0.00145

Test Item

0.70340

0.69645

0.68645

0.69543

0.00852

Blank OD Value (mean of 6 replicate values): 0.04240

True OD value = OD Raw – OD Blank

Table 3. Individual Tissue Viability of Epidermis Units (Relative)

 

% Individual Viability

R1

R2

R3

Mean

SD

Positive control

7.02

6.97

6.63

6.87

0.21

Test Item

101.33

100.33

98.89

100.19

1.23

Applicant's summary and conclusion

Interpretation of results:
other: No category (CLP Regulation EC no. 1272/2008)
Conclusions:
Under in vitro RHE test method performed in Episkin model the test item is predicted to be non-irritant.

Executive summary:

An in vitro skin irritation test of the test item was performed in a reconstructed human EpiSkin TM model, according to OECD TG 439 (GLP study). Three epidermis units were treated with 10 μL test item for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with PBS. The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol, incubating the tissues at room temperature protected from light for 4 hours, and finally measuring the concentration of formazan by determining the OD at 570 nm. Under the test conditions, the mean percent viability of the treated tissues was 100.19%, versus 6.87% of the positive control (5% Sodium Dodecyl Sulfate) and 100% of the negative control (PBS). Therefore, the test item is predicted to be non-irritant.