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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
Study period:
1 (reliable without restriction)

Data source

Reference Type:
study report

Materials and methods

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

Test material

Chemical structure
Reference substance name:
Octadecanoic acid, reaction products with triethylenetetramine, chloromethane-quaternized
EC Number:
EC Name:
Octadecanoic acid, reaction products with triethylenetetramine, chloromethane-quaternized
Cas Number:
Molecular formula:
Not applicable - UVCB substance
Octadecanoic acid, reaction products with triethylenetetramine, chloromethane-quaternized
Test material form:

In vitro test system

Test system:
human skin model
Source species:
Cell type:
non-transformed keratinocytes
Details on test system:
The test was carried out with the reconstituted three-dimensional human skin model EpiDerm. This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts.

The EpiDerm epidermis model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous and granular layers and a multi-layered stratum corneum analogous to patterns found in vivo.

To check the non-specific MTT-reducing capability of the test item 25 mg of the test item were mixed per 1 mL MTT medium and incubated for 60 min at 37 ± 1 °C in the incubator.
The mixture did not turn blue/purple. Thus, the additional test with freeze-killed tissues and the quantitative corrections were not necessary.

After pre-incubation, the tissues were treated with each dose group in triplicate, starting with the negative control. Start time was recorded with dosing of the first tissue and occurred sequentially for the other tissues, e.g. in one-minute intervals. After dosing of all tissues, all plates were incubated for 35 ± 1 min. Afterwards all plates were removed from the incubator and placed under the sterile flow for the remaining time until the 60 ± 1 min incubation time of the first dosed tissue was over.

Then the tissues were washed with DPBS. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. The inserts were placed in prepared new 6-well plates containing 0.9 mL pre-warmed fresh assay medium per well and the tissue surface was dried using a sterile cotton tip. The plates were post-incubated at 37 ± 1 °C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing 0.9 mL fresh assay medium and incubated for additional 18 ± 2 h.

After this post-incubation period the bottom of the inserts were blotted on sterile blotting paper and the inserts were transferred in a prepared 24-well plate containing 300 μL pre-warmed MTT medium. This plate was incubated for 3 h ± 5 min at 37 ± 1 °C, 5.0% CO2, humidified to 95%.
After the MTT incubation period, the tissues were rinsed three times with DPBS and afterwards placed on blotting paper to dry. The tissues were transferred into 12-well plates and immersed in 2 mL isopropanol. Extraction was carried out protected from light at room temperature for at least 2 h with gentle shaking.

The extract was pipetted up and aliquots per each tissue were transferred into a 96-well plate. OD was measured at 570 nm with a filter band pass of maximum ± 30 nm without reference wavelength in a plate spectrophotometer using isopropanol as a blank.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
25 mg
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:

Results and discussion

In vitro

Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Octadecanoic acid, reaction product with triethylenetetramine, chloromethane-quaternized was applied topically to the EpiDerm tissue for 60 min followed by a 42 h post-incubation period.
The controls confirmed the validity of the study.
Negative controls validity:
Positive controls validity:

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (102.5%) after 60 min treatment and 42 h post-incubation.

The controls confirmed the validity of the study. The mean absolute OD570 of the three negative control tissues was >= 0.8 and ≤ 2.8 (1.798). The mean relative tissue viability (% negative control) of the positive control was <= 20% (3.5%). Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.5% - 6.5%).

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