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Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
March 19th to July 20th, 2018
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:
2018

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
Principles of method if other than guideline:
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 Function
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
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethyl 4-oxovalerate
EC Number:
208-728-2
EC Name:
Ethyl 4-oxovalerate
Cas Number:
539-88-8
Molecular formula:
C7H12O3
IUPAC Name:
ethyl 4-oxopentanoate

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:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM tissue kit - EPI-200-SIT
- Tissue batch number(s): (1) 25887, (2) 28600, (3) 28634
- Delivery date: (1) 20. Mar. 2018, (2) 24. Apr. 2018, (3) 17. Jul. 2018
- 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.

SOLUTIONS AND MEDIA
- MTT solution: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (=MTT), which 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)
- Assay Medium: serum-free DMEM (Dulbecco’s Modified Eagle’s Medium)
- DPBS-buffer: solution for the rinsing of the tissues and solvent for MTT concentrate, also used as negative control. Composition prepared: KCl 0.2 g, KH2PO4 0.2 g, NaCl 8.0 g, Na2HPO4 * 7H2O 2.16 g, H2O ad 1 litre. Composition of the subset procured: KCl 0.2 g, KH2PO4 0.2 g, NaCl 8.0 g, Na2HPO4 * 2H2O 1.44 g, H2O ad 1 litre. The buffer which was procured was used as negative control and for rinsing the test item from the tissues. The buffer which was prepared was used as solvent for MTT concentrate and for rinsing the outside of the inserts at the end of the incubation time with MTT.

PRE-TESTS
- 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.
- 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.

MAIN TEST
-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 and 55 minutes (in all experiments).
- 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 % SDSsolution, 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. 25 minutes 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 with DPBS 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). 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 25 minutes (1. and 2. experiment) and for 23 hours 20 minutes (3. experiment) 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 15 minutes (1.experiment), 19 hours 20 minutes (2.experiment) and 19 hours 5 minutes (3.experiment) for post-incubation at 37 ± 1 °C and 5.0 ± 0.5 % CO2.
- MTT Assay: after a total incubation time of 42 hours 40 minutes (1.experiment), 42 hours 45 minutes (2.experiment) and 42 hours 25 minutes (3.experiment), 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 thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 μl solution (each) were pipetted into a 96-wellplate which was read in a plate spectrophotometer at 570 nm.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant/corrosive to skin if the viability after the exposure is less than or equal to 50 %
- The test substance is considered to be non-irritant to skin if the viability after the exposure is greater than 50 %.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 30 μl

NEGATIVE CONTROL
- Amount(s) applied: 30 μl

POSITIVE CONTROL
- Amount(s) applied: 30 μl
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
2 hours 40 minutes (1st experiment), 42 hours 45 minutes (2nd experiment) and 42 hours 25 minutes (3rd experiment)
Number of replicates:
three

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1st experiment
Value:
37.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2nd experiment
Value:
50.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3rd experiment
Value:
28.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Skin Irritation Potential of the Test Item: the mean value of relative tissue viability of the test item was reduced to 37.1 % (1st experiment), 50.5 % (2nd experiment) and 28.9 % (3rd experiment). Two of the three results are below the threshold for skin irritation potential (50 %). Test items that induce values below the threshold of 50 % are considered at least irritant to skin.

- OTHER EFFECTS:
- Direct-MTT reduction: direct MTT reduction by the test item had not taken place and no data correction was necessary.
- Colour interference with MTT: no colour interference

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. OD demanded 0.8-2.8. 1st experiment: OD= 1.3; 2nd experiment: OD = 1.2; 3rd experiment: OD = 1.6
- Acceptance criteria met for positive control: yes. % tissue viability of positive control demanded 20 % tissue viability of negative control control. 1st experiment: 2.5 %; 2nd experiment: 4.1%; 3rd experiment: 3.1 %.
- Acceptance criteria met for variability between replicate measurements: SD of mean viability of the tissue replicates demanded 18 %. 1st experiment: 11.0 % (negative control), 0.0 % (positive control), 16.7 % (test item); 2nd experiment: 9.8 % (negative control), 0.2 % (positive control), 6.7 % (test item); 3rd experiment: 8.8 % (negative control), 0.0 % (positive control), 3.2 % (test item).
- Range of historical values if different from the ones specified in the test guideline: values for negative control and for positive control were within the range of historical data of the test facility
- Validity and Acceptability: all validity criteria were met. Three experiments were performed. The result of the first test run was equivocal, because two of the three replicates of the test item were below the threshold of 50% and one replicate was above 50 %. The test item showed skin irritation potential. But as the result was equivocal, a second test run was performed. Therefore, the experiments are considered valid according to the validity criteria. The result of the second test run was equivocal, too, because two of the three replicates of the test item were below the threshold of 50% and one replicate was above 50 %. The test item showed no skin irritation potential. But as the result was equivocal, a third and final test run was performed. The result of the third test run was unequivocal and the test item showed skin irritation potential. The three test runs together lead to the final classification that the test item has at least skin irritation potential.

Applicant's summary and conclusion

Interpretation of results:
other: the substance should be classified according to the CLP Regulation (EC) No.1272/2008 in Category 1 or Category 2
Conclusions:
The substance is a skin irritant (Cat.2) or a skin corrosive (Cat.1)
Executive summary:

The potential of the substance to evoke skin irritation was evaluated in vitro in a reconstructed human epidermis (RhE) test method conducted according to the OECD Guideline 439 and EU Method B.46. The human reconstructed epidermis model was topically exposed to the neat test item, followed by a cell viability test. Cell viability was measured by dehydrogenase conversion of MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazoliumbromide, present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percentage reduction of cell viability in comparison of untreated negative controls was used to predict the skin irritation potential. Sodium dodecyl sulphate was tested as a positive control.

All validity criteria were met. Three experiments were performed. The result of the first test run was equivocal, because two of the three replicates of the test item were below the threshold of 50 % and one replicate was above 50 %. In summary, the test item showed skin irritation potential. But as the result was equivocal, a second test run was performed. The result of the second test run was equivocal, too, because two of the three replicates of the test item were below the threshold of 50 % and one replicate was above 50 %; the test item showed no skin irritation potential. But as the result was equivocal, a third and final test run was performed. The result of the third test run was unequivocal and the test item showed skin irritation potential. The three test runs together lead to the final classification that the test item has at least skin irritation potential. After the treatment, the mean value of relative tissue viability was reduced to 37.1 % (1st experiment), 50.5 % (2nd experiment) and 28.9 % (3rd experiment). Two of the three results are below the threshold for skin irritation potential (50 %).

The test substance is considered at least skin irritant.

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