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skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 28 January to 30 June 2022
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
conducted under GLP conditions

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)
See 'Any other information on materials and methods incl. tables' section
GLP compliance:
yes (incl. QA statement)

Test material

impurity 1
Chemical structure
Reference substance name:
Cas Number:
Molecular formula:
impurity 2
Chemical structure
Reference substance name:
Cas Number:
Molecular formula:
Constituent 1
Chemical structure
Reference substance name:
Cas Number:
Molecular formula:
Test material form:

In vitro test system

Test system:
human skin model
Source species:
Cell type:
non-transformed keratinocytes
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
unchanged (no vehicle)
Details on test system:
The EpiDerm'M Model (EPl-200) (MatTek Corporation, Ashland, USA) consists of normal, humanderived epidermal keratinocytes which have been cultured to form a multilayered, 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 arranged in patterns analogous to those found in vivo. The EpiDerm'M Model incorporates several features which make it advantageous in the study of potential dermal toxicity. First, the test system uses a serum-free medium which eliminates the possibility of serum protein and test article interaction (Shopsis and Eng, 1988). Secondly, the target cells are epithelial, derived from human skin. Third, since the tissue has a functional stratum corneum, the test materials are applied directly to the tissue surface, at air interface, so that undiluted and/or end use dilutions can be tested directly. Prior to use, each 6-, 24- and 96-well plate will be uniquely identified with a number written in permanent marker with the test article identification or control treatment group.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Negative control: Neat (undiluted)
Positive control: 5%
Test item: Neat (undiluted)
Duration of treatment / exposure:
60 +/- 1 minutes
Duration of post-treatment incubation (if applicable):
42 +/- 2 hours post-exposure incubation period
Number of replicates:
Negative control: 3
Positive control: 3
Test item: 3

Results and discussion

In vitro

Irritation / corrosion parameter:
% tissue viability
Vehicle controls validity:
not examined
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The assay was accepted since the following criteria were met:
1) the positive control (5% SDS) resulted in a mean tissue viability <20% (2.63%);
2) the mean OD570 value of the negative control tissues (1.637) was >= 0.8 and <= 2.8;
and 3) the standard deviation of the positive and negative control calculated from individual percent tissue viabilities of the three identically treated replicates were <= 18% (6.63% for the negative control and 0.19% for the positive control).

Any other information on results incl. tables

The skin irritancy potential of the test article was evaluated as described in OECD TG 439:

In VitroResultGHS Category
mean tissue viability ≤ 50%Category 1 or 2*
mean tissue viability > 50%No Category

* - Additional testing would be warranted to resolve between GHS Category 1 (Severe/Corrosive) and GHS Category 2 (Irritant)


Test results:

 Test Article Number Sponsor’s Designation Conc. (w/v) pH Mean Viability (%) Skin Irritation Prediction
 22AA20GR-87-6331 (Scentaurus Melrose)Neat5.0103.25 +/- 8.93Non-Irritant
 Positive Control SDS 5% NA 2.63 +/- 0.19


Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
According to the prediction model presented in OECD TG 439, the test article was predicted to be non-Irritant (GHS No Category) since the treated tissues resulted in a mean tissue viability of
Executive summary:

The EpiDerm™ Skin Model (MatTek Corporation, MA, USA) was used to assess the potential dermal irritation of the test article. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) conversion assay, which measures the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, the succinate dehydrogenase reduction of MTT) to a blue formazan precipitate, was used to assess cellular metabolism after exposure to the test article. The protocol was based upon the OECD guideline, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (TG 439).


According to the prediction model presented in OECD TG 439, the test article was predicted to be non-Irritant (GHS No Category) since the treated tissues resulted in a mean tissue viability of 103.25%.