Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
OECD Guideline for the Testing of Chemicals, Version 439, adopted 28. July 2015, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method”
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
GLP-Conformity All procedures will be according to the principles of GLP (Chemikaliengesetz §19a and §19b and annexes 1 and 2 from 28. Aug. 2013, published in Federal Law Gazette, Germany (BGBl) No. 55/2013 as of 06. Sep. 2013, and further revisions).

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
additive
Test material form:
liquid
Details on test material:
- State of aggregation: not applicable
- Activation: not required
water as a additive
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: The test material is representative of the registered substance
- Expiration date of the lot/batch: not relevant
- Purity test date: not relevant

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none

In vitro test system

Test system:
isolated skin discs
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: MatTek In Vitro Life Science Laboratories, Bratislava
Source strain:
not specified
Details on animal used as source of test system:
SOURCE ANIMAL
- Source: Homo sapiens
- Sex: unknown
- Age at study initiation (in days): unknown
- Weight at study initiation: unknown
- Housing: unknown
- Diet (e.g. ad libitum): unknown
- Water (e.g. ad libitum): unknown
- Acclimation period: not required
Justification for test system used:
protection of animals
Vehicle:
other: DPBS buffer
Details on test system:
Specification of test material of 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
Day of delivery: 13. Feb. 2018
Batch no.: 25878
Chemicals and test vessels
MTT
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (=MTT) can be reduced to a blue formazan.
A MTT stock solution of 5 mg/mL in DPBS buffer was prepared and stored in aliquots of 2 mL in the freezer (– 20 ± 5 °C).
2 mL of the stock solution were thawed and diluted with 8 mL of medium. This MTTsolution with the resulting concentration of 1 mg/mL was used in the test.
For the pre-test (testing the ability of direct MTT reduction), the stock solution was thawed and diluted with serum-free MEM directly before use.
For the main test, the stock solution was thawed and diluted with assay medium directly before use.
MEM with Phenol Red for Pre-Test
Serum-free MEM (Minimum Essential Medium), procured by Life Technologies GmbH, batch no.: 1853467
Assay Medium
Serum-free DMEM (Dulbecco’s Modified Eagle’s Medium), procured by MatTek In Vitro Life Science Laboratories, batch no.: 020818ALE
Isopropanol
CH3-CH(OH)-CH3, p.A., 99.9 %, batch no.: 276245555, used as extracting solvent for formazan
DPBS-buffer
Solution for the rinsing of the tissues and solvent for MTT concentrate, also used as negative control. A subset was procured by MatTek In Vitro Life Science Laboratories; the other subset was prepared by LAUS GmbH.
Composition of the subset from MatTek In Vitro Life Science Laboratories (batch no.: 092817MGKA):
KCl 0.2 g
KH2PO4 0.2 g
NaCl 8.0 g
Na2HPO4 * 7H2O 2.16 g
H2O ad 1 L
Composition of the subset from LAUS GmbH (batch no.: 20171114):
KCl 0.2 g
KH2PO4 0.2 g
NaCl 8.0 g
Na2HPO4 * 2H2O 1.44 g
H2O ad 1 L
The buffer which was procured by MatTek Corporation was used as negative control and for rinsing the test item from the tissues. The buffer which was prepared by LAUS GmbH was used as solvent for MTT concentrate and for rinsing the outside of the inserts at the end of the incubation time with MTT.
Test Vessels
All vessels used are made of glass or sterilised plastic. The glassware was sterilised before use by autoclaving. The following vessels were used:
6-well-plates
24-well plates
96-well-plate

Calculation
Calculations were performed as follows:
• Calculation of mean OD of the blank isopropanol (ODBlk)
• Subtraction of mean ODBlk of each value of the same experiment (corrected values)
• Calculation of mean OD of the two replicates for each tissue
• Calculation of mean OD of the three relating tissues for controls and test item

Note: Corrected OD value of negative control corresponds to 100 % viability

The photometric absorbance of the negative controls is considered as 100%. For each replicate of test item and positive control, tissue viability is calculated as % photometric absorbance compared with the mean of the negative controls:

% tissue viablity = 100 * OD replicate test positive control/OD Mean of negative control

1 OD = Optical Density

Assessment
Skin irritation potential of the test item is assessed as given in the following table:
Table 8.2 a Assessment of Skin Irritation Potential
% Tissue viability Assessment UN GHS classification
≤ 50 % of negative control Corrosive/ Irritant to skin UN GHS Category 1 or 2
> 50 % of negative control Non-irritant to skin No Category for Skin Irritation
Control samples:
yes, concurrent negative control
Amount/concentration applied:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: The test material is representative of the registered substance, and no significant differences in the purity profile exist
- Expiration date of the lot/batch: not relevant (stable)
- Purity test date: not relevant, the test material is representative of the registered substance

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was stored in the test facility in a closed vessel at room temperature (20 ± 5°C).
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble
- Reactivity of the test substance with the solvent: none (contains water)

NEGATIVE CONTROL
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 ± 0.5% CO2 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.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: none
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
24 h + 19 h (see Details of study design)
Number of replicates:
For each treatment group (negative control, test item and positive control) a 6-well-plate was prepared with 0.9 mL assay

Test animals

Species:
other: in vitro
Strain:
other: not applicable
Details on test animals and environmental conditions:
not applicable

Test system

Type of coverage:
other: in vitro
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
yes
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
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 were 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 ± 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 25 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 min-utes (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 19 hours for post-incubation at 37 ± 1°C and 5.0 ± 0.5% CO2.
Duration of treatment / exposure:
24 h
Observation period:
42 h after finalization of incubation tests on viability were started
After a total incubation time of 42 hours 25 minutes, a 24-well-plate was prepared with 300 µL freshly prepared MTT-solution 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 at 37 ± 1°C and 5.0 ± 0.5% CO2.
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 thor-oughly 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.
Number of animals:
0
Details on study design:
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 ± 0.5% CO2 for 1 hour.
The resulting solution was colourless, therefore no binding capacity had to be tested.

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 ± 0.5% CO2 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 ± 0.5% CO2 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 ± 0.5% CO2 for 18 hours 55
minutes.

LITERATURE
The study was conducted in accordance with the following guidelines:
• OECD Guideline for the Testing of Chemicals, Version 439, adopted 28. July 2015, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method”
• Commission Regulation (EU) No. 640/2012 amending Regulation (EC) No. 761/2009, Annex III, EU method B.46 “IN VITRO SKIN IRRITATION: RECON-STRUCTED HUMAN EPIDERMIS MODEL TEST”, adopted 06. Jul. 2012

Additional information was taken from:
• ECVAM international validation study on in vitro tests for acute skin irritation: “Report on the validity of the EPISKIN and EpiDerm assays and on the Skin Integrity Func-tion Test” (Altern Lab Anim. 2007 Dec; 35 (6): 559-601).
• Protocol for In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT), Rev. 07/11/2014, MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, Bratislava - Slovakia

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Replicates: Three tissues of the human skin model EpiDermTM were treated with the test item for 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).
Value:
ca. 0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
Exacta data not avvalable at the moment
Other effects / acceptance of results:
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 values was within the required
acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.1.
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 itemwas acceptable (required: ≤ 18%).

Applicant's summary and conclusion

Interpretation of results:
Category 1A (corrosive) based on GHS criteria
Remarks:
clear effect, similar to positive controls
Executive summary:

In a in vitro test according to OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method) with 3 tissues of the human skin model EpiDermTM treated with the test item for 60 minutes, the relative tissue viability was significantly reduced. Test items that induce values below the threshold of 50% are considered at least irritant to skin.