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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11. Feb. 2019 - 15. May 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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
The pre-incubation time was 1 hour, instead of 18 ± 3 hours overnight. This can be seen as uncritical, because the pre-incubation time must be at least 1 hour (in consultation with the tissue supplier; MatTek In Vitro Life Science Laboratories).
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethoxycarbonylmethyl ethyl phthalate
EC Number:
201-555-3
EC Name:
Ethoxycarbonylmethyl ethyl phthalate
Cas Number:
84-72-0
Molecular formula:
C14H16O6
IUPAC Name:
ethyl 2-[(2-ethoxy-2-oxoethoxy)carbonyl]benzoate
Test material form:
liquid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDermTM, MatTek In Vitro Life Science Laboratories, Bratislava
Details on animal used as source of test system:
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.
Justification for test system used:
This Test Guideline addresses the human health endpoint skin irritation. It is based on the in vitro test system 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. The RhE test system uses human derived non-transformed keratinocytes as cell source to reconstruct an epidermal model with representative histology and cytoarchitecture. Performance Standards are available to facilitate the validation and assessment of similar and modified RhE-based test methods, in accordance with the principles of Guidance Document No. 34.
Vehicle:
unchanged (no vehicle)
Details on test system:
Pre - Tests
- Nylon mesh compatibility
- Assessment of Interference of Coloured or Staining Test Items
It is tested whether the test item develops a colour without MTT addition. 30 µL test item is 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.
- Assessment of Direct Reduction of MTT by the Test Item
30 µL test item is added to 1 mL of MTT solution and the mixture is incubated in the dark at 37 ± 1°C, 5.0 ± 1% CO2 and ≥ 95% relative humidity for 1 hour. Untreated MTT medium is used as control.

Pre-Incubation of Tissues
All working steps are performed under sterile conditions.
The viable tissues are transferred into the medium containing wells by using sterile forceps 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 (lower row) are filled with fresh assay medium (0.9 mL). Every tissue is transferred subsequently into a well of the lower row. All 6-well-plates are set into the incubator at 37 ± 1°C, 5.0 ± 1% CO2 and ≥ 95% relative humidity for 1 hour.

Treatment
3 tissues are used as negative control (treated with 30 µL DPBS buffer), a nylon mesh is added in order to ensure sufficient contact with the tissue surface.
3 tissues are used as positive control (treated with 30 µL 5% SDS-solution), a nylon mesh is added in order to ensure sufficient contact with the tissue surface.
3 tissues are used for treatment with the test item:
30 µL test item are applied, and a nylon mesh is added in order to ensure sufficient contact with the tissue surface.
Tissues are dosed in 1-minute-intervals. After dosing the last tissue, all plates Are transferred into the incubator for 35 minutes at 37 ± 1°C, 5.0 ± 1% CO2 and ≥ 95% relative humidity.
1 hour after the first application, the inserts Are removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals.
After rinsing thoroughly with DPBS, each tissue Is 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 Is then carefully dried with a sterile cotton tipped swab.
Then, the tissues Are set in the incubator for 22 hours at 37 ± 1°C, 5.0 ± 1% CO2 and ≥ 95% relative humidity.

After a total incubation time of 38 hours and 50 minutes, a 24-well-plate Is prepared with 300 µL freshly prepared MTT-solution (1 mg/ml) in each well. MTT Assay is performed and plates are read in a plate spectrophotometer at 570 nm.

Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
30 µL of the test item
Duration of treatment / exposure:
After dosing the last tissue, all plates were transferred into the incubator for 35 minutes at 37 ± 1°C, 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.
Duration of post-treatment incubation (if applicable):
total post-incubation time: 38 hours and 50 minutes
Number of replicates:
- 3 replicates per test item
- 3 replicates negative controls,
- 3 replicates positive controls
- 8 replicates blank isopropanol (OD 570 nm) for absorbance measurement

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Test item (one experiment)
Value:
100.6
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Criteria
- OD of negative control: ≥ 0.8 and ≤ 2.8
- % tissue viability of positive control SDS: ≤ 20% of negative control
- SD of mean viability of the tissue replicates (%): ≤ 18%
All criteria were met.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item Ethylphthalyl ethyl glycolate is considered as non- irritant to skin.
Executive summary:

Determination of Skin Irritation Potential of Ethylphthalyl ethyl glycolate in the Reconstructed human Epidermis (RhE) Test Method following EU-Method B.46 and OECD 439:

One valid experiment was performed.

Tissue of the human skin model EpiDermTMwas treated with the test item (undiluted) for 60 minutes. Three replicates were used for chemical treatment and for controls, respectively.

The test item was applied directly to each tissue and spread to uniformly cover the tissue surface (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 of 0.8 ≤ mean OD ≤ 2.8, OD was 1.8. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 3.0 % (required: ≤ 20%).

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 100.6 %. This value is above the threshold for skin irritation potential (50%). Test items that induce values above the threshold of 50% are considered non- irritant to skin.

Therefore, the test itemEthylphthalyl ethyl glycolateis considered non- irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.