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Diss Factsheets

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 6th, 2019 to May 10th, 2019
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
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Adopted July 28th, 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Adopted July, 6th, 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Hexadecyl hydrogen maleate
EC Number:
220-942-8
EC Name:
Hexadecyl hydrogen maleate
Cas Number:
2944-06-1
Molecular formula:
C20H36O4
IUPAC Name:
(2Z)-4-(Hexadecyloxy)-4-oxo-2-butenoic acid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SYSTEM SPECIFICATION:
The test system is a commercially available EpiDermTM-Kit, procured by MatTek In Vitro Life Science Laboratories, Bratislava.
Designation of the kit: EPI-200-SIT
Day of delivery: May 7th, 2019
Batch no.: 28695.
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.

CHEMICALS AND MEDIA:
- MTT: 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 MTT-solution 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) was procured by Life Technologies GmbH, batch no.: 2033578.
- Assay Medium: serum-free DMEM (Dulbecco’s Modified Eagle’s Medium) was procured by MatTek In Vitro Life Science Laboratories, batch no.: 050219MSC.
- Isopropanol: CH3-CH(OH)-CH3, p.A., 99.9 %, batch no.: 457264070 was used as extracting solvent for formazan .
- DPBS-buffer: DPBS-buffer solution was used for the rinsing of the tissues, solvent for MTT concentrate and also used as negative control. A subset was procured by MatTek In Vitro Life Science Laboratories; the other subset was prepared in the testing facility. Composition of the subset from MatTek In Vitro Life Science Laboratories (batch no.: 031219MSA): 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 prepared in the testing facility (batch no.: 20181022): 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 the testing facility was used as solvent for MTT concentrate and for rinsing the outside of the inserts at the end of the incubation time with MTT.

PERFORMANCE OF TH STUDY:
The test item was ground to a fine powder before usage.
- Pre – Tests:
It was tested whether the test item develops a colour without MTT addition. 27.4 mg of the test item were given in a test tube with 0.3 mL H2O demin. and incubated at 37 ± 1°C, 5.0 ± 1% CO2 and ≥ 95% relative humidity for 1 hour. The resulting solution was colourless, therefore no binding capacity had to be tested. The ability of the test item to directly reduce MTT was tested. 25.9 mg of the test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at 37 ± 1°C, 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. After arrival, the tissues were inspected for viability. Then, the viable tissues were transferred into the wells with the assay medium and placed into the incubator at 37 ± 1°C, 5 ± 1% CO2 and ≥ 95% relative humidity for 1 hour. After 1 hour pre-incubation, the other 3 wells of each plate 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, 5.0 ± 1% CO2 and ≥ 95% relative humidity for 19 hours and 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: replicate 1 = 26.1 mg, replicate 2 = 25.8 mg, replicate 25.3 mg. The tissues were wetted with 25 µL DPBS buffer before applying the test item and spreading it to match the tissue size. Tissues were dosed in 1-minute-intervals. During the application of the second replicate of the test item the medium had to be removed, because a small amount of test item dropped into the medium. Then the well was refilled with 900 µL fresh medium. 24 ± 1 min. 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, 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, 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, ≥ 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:
Replicate 1 = 26.1 mg
Replicate 2 = 25.8 mg
Repicate 3 = 25.3 mg
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
41 hours and 57 minutes
Number of replicates:
Three

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
One
Value:
138.2
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
SD = 4.8%
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: none

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The validity of the skin irritation study 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:
- Acceptance criteria met for negative control: yes. OD of negative control: 1.5
- Acceptance criteria met for positive control: yes. % tissue viability of positive control SDS: 3.9
- Acceptance criteria met for variability between replicate measurements: 9.5 % (negative control), 0.8 % (positive control), 4.8 % (test item).
- Range of historical values: negative and positive controls are in the range of historical values.

Applicant's summary and conclusion

Interpretation of results:
other: Not classified for skin irrittaion according to CLP criteria
Conclusions:
According to the in vitro results and OECD 439 criteria, the test item Hexadecyl hydrogen maleate is considered as non-irritant to skin.
Executive summary:

The in vitro study was performed in order to evaluate the potential of Hexadecyl hydrogen maleate to evoke skin irritation in a reconstructed human epidermis (RhE) test method. The Test Method was performed following EU-Method B.46 and OECD 439. One valid experiment was performed. 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). 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 (1.5). The positive control showed clear irritating effects. The mean value of relative tissue viability was indeed reduced to 3.9% (required: ≤ 20%) with the positive control. 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 138.2 %. This value is above the threshold for skin irritation potential (50%). Therefore, the test item Hexadecyl hydrogen maleate is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.