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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Toxicological information

Skin irritation / corrosion

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Administrative data

skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study according to guideline and GLP

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
C39 H29 F3 O2
Details on test material:
- Name of test material (as cited in study report): Ligand TFME-DPP
- Physical state: solid, white
- Batch No.: 01829-00216
- pH: Ca. 5 (undiluted test substance, moistened with water)
- Storage condition of test material: at room temperature
- Homogeneity: Homogenous by visual inspection

Test animals

other: not applicable (in vitro test)
other: not applicable (in vitro test)

Test system

Type of coverage:
other: not applicable (in vitro test)
Preparation of test site:
other: not applicable (in vitro test)
physiological saline
other: not applicable (in vitro test)
Amount / concentration applied:
negative control tissues: 30 ul PBS
positive control tissues: 30 ul 5% SDS
test tissues: 25 ul PBS + 25 ul solid test material
Duration of treatment / exposure:
1 hour (25 min at room temperature + 35 min at 37°C), then tissues were washed with sterile PBS, transferred into fresh medium and dried with a sterile cotton swab
Observation period:
42 hours (2nd medium change at 24 +/- 2 hours after exposure)
Number of animals:
not applicable (in vitro test)
For negative control, positive control and test substance, three tissues each were employed. Additionally, one killed tissue each was used for the test substance and negative control in order to detect direct MTT reduction
Details on study design:
The EpiDerm(TM) Skin Irritation Test is based on the finding that chmical-induced skin irritation is the result of a cascade of events that is initiated by penetration of the chemicals through the stratum corneum where they may damage the underlying layers of keratinocytes and other skin cells. The test method used here measures the initiating event in the cascade, i.e. cell / tissue damage. This is accomplished via the means of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. Living cells convert MTT enzymatically into blue formazan salt which is quantitatively measured after extraction from tissues.
EpiDerm(TM) consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. Based on the results of a validation study, it was concluded by EVCAM that the EpiDerm(TM) human epidermis model is suitable to be used for distinguishing between irritant and non-irritant chemicals.

To determine whether Ligand TFME-DPP constitutes a skin irritant, EpiDerm tissues were preincubated for 16-22 hours in 0.9 mL medium and subsequently treated with the test substance, the positive control or the negative control. For test substance treatment, 25 ul of PBS were added first. Thereafter, a bulk volume of 25 ul of the solid test material (ca. 12 mg) was applied with a sharp spoon and homogeneously distributed together with the fluid. Control tissues were concurrently applied with 30 ul of sterile PBS (negative control, NC) or with 30 ul of 5 % SDS (positive control, PC). A nylon mesh was placed carefully onto the tissue surface of the NC and PC controls afterwards.
Following incubation for 25 min at room temperature and 35 min at 37°C, the tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted onto sterile absorbent paper and transferred into new 6-well-plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. The tissues were then incubated at 37°C for 24 +/- 2 hours, subsequently transferred into new 6-well plates pre-filled with 0.9 mL fresh medium and incubated at 37°C for additional 18 +/- 2 hours.
The medium was then replaced by 0.3 mL MTT solution and the tissues were incubated at 37°C for 3 hours. They were then washed with PBS to stop the MTT-incubation. The formazan salt was extracted with isopropanol from the tissues and the optical density was determined spectrophotometrically. The quotient of test tissue absorption divided by negative control absorption was used to calculate tissue viability.

Results and discussion

Any other information on results incl. tables

Preliminary findings indicated that the test substance can reduce MTT directly. Therefore, killed control tissues treated as negative control were performed in parallel. However, the result of the killed control did not indicate an increased MTT reduction, therefore the killed control was not used for viability calculation.

The EpiDerm(TM) in vitro skin irritation test showed no reduction in cell viability after treatment with test substance compared to negative control (115% and 100%, respectively). In contrast, mean viability of the positive control was 4%.

     tissue 1  tissue 2  tissue 3  mean  SD
 Negative control (NC)    mean OD(570)  2.160  2.206 2.317  2.228  
 viability (% of NC)  97.0  99.0  104.0  100  3.62
 Test substance     mean OD(570)  2.608  2.645  2.420  2.558  
 viability (% of NC)  117.1  118.7  108.7  115  5.4
 Positive control     mean OD(570)  0.082  0.081  0.072  0.079  
 viability (% of NC)  3.7  3.7  3.2  4  0.25

Applicant's summary and conclusion