Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 February 2018 to ****
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
-Purity: >95% (nominal); UVCB
-Description: Amber Liquid
- Storage: room temperature, in the dark

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: reconstructed human epidermis tissues
Cell source:
other: Not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test item was found to have the potential to directly reduce MTT and therefore, as a precaution, additional non-viable tissues were incorporated into the testing for correction purposes. The test item was found to cause color interference with the MTT endpoint therefore, additional tissues were incorporated into the testing to correct for this. A third set of controls was included, comprising non-viable tissues, to prevent a double correction from a colored test item that also reduces MTT. At the end of the post exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre labeled micro tubes and stored in a freezer for possible inflammatory mediator IL-1α determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre labeled 96 well plate. The optical density was measured at 570 nm (OD570).
Control samples:
other: Negative Control: Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++ and Positive Control: Sodium Dodecyl Sulphate (SDS)
Amount/concentration applied:
10 µL (26.3 µL/cm2) of the test item was applied to the epidermis surface.
Duration of treatment / exposure:
Triplicate tissues were treated with the test item for an exposure period of 15 minutes
Duration of post-treatment incubation (if applicable):
At the end of the exposure period each tissue was rinsed before incubating for 42 hours.
Number of replicates:
Three

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post exposure incubation period of 42 hours
Value:
133.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Any other information on results incl. tables

Test for Direct MTT Reduction

The MTT solution containing the test item turned purple which indicated that the test item directly reduced MTT. Therefore, an additional procedure using water-killed tissues was performed. However, the results obtained showed that no effects on OD570values that indicated the test item was totally rinsed off with no residual test item remained on or in the tissues. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results.

Test Item, Positive Control Item and Negative Control Item

The relative mean viability of the test item treated tissues was 133.9% (>50%) after a 15‑minute exposure period and 42‑hour post‑exposure incubation period.

Assessment of Color Interference with the MTT endpoint

The test item was found to produce a colored solution which might interfere with the MTT endpoint. Therefore, color correction tissues were incorporated into the test to correct for this possibility. These tissues were treated identically to the tissues of the main test with the exception of being placed into assay medium for 3 hours post exposure instead of MTT. Three tissues were dosed with the test item and three remained untreated to act as negative controls.

Quality Criteria

The relative mean viability for tissues treated with 5% (w/v) SDS aqueous solution (positive control) was 2.7% relative to the negative control treated tissues and the standard deviation value of the viability was 0.6% (≤18%). The positive control acceptance criteria were therefore satisfied.

The mean OD570for the negative control (DPBS) treated tissues was 0.736, within the range of 0.6 - 1.5 and the standard deviation value of the viability was 1.2% (≤18%). The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 7.1% (≤18%). The test item acceptance criterion was therefore satisfied.

However, the interference of the remained test item on the viable tissues with the MTT endpoint could not be corrected because the amber colored test item adhered to remained on the viable tissue surfaces, but not on the non-viable tissue surfaces after rinsing and the precise tissue viability could not be obtained.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Although the precise tissue viability could not be obtained, the relative mean viability of the test item treated tissues was 133.9%, well above 50% that would require classification for skin irritation.
Executive summary:

The test item, Fatty acids, C18-unsatd., dimers, compds, with 4,5-dihydro-2-nortall-oil alkyl-1H-imidazole-1-ethanol and tall-oil fatty acids, was amber color that interfered with the MTT endpoint of this assay. Additionally the test item adhered to and remained on the culture surfaces of the viable tissues, but not on the non-viable tissues after rinsing. Therefore, the interference of the remained amber color test item on the viable tissues with the MTT endpoint could not be corrected with non-viable tissues. Although the precise tissue viability could not be obtained, the relative mean viability of the test item treated tissues was 133.9%, well above 50% that would require classification for skin irritation. In addition, the score of the test item in the BCOP assay was 0.2, well below the score of 3 that would require classification for eye irritation. The undiluted test item had no signs of local skin irritation to mouse ears in the LLNA in mice. The acute oral toxicity study of the test item showed no clinical signs of toxicity, no gross lesions at necropsy, and acceptable weight gain by all animals dosed by gavage at 2000 mg/kg. Therefore, the existing data provide sufficient weight of evidence to justify not classifying the test item for dermal irritation in accordance to Regulation (EC) No. 1272/2008 Classification, Labelling and Packaging of Substances and Mixtures (EU CLP) and United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS).