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Skin irritation / corrosion

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skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13th May 2019 to 28th May 2019
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
according to guideline
other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.46 “In vitro Skin Irritation: Reconstructed Human Epidermis Model Test ". Official Journal of the European Union No. L142; Amended by EC No.
GLP compliance:

Test material

Reference substance name:
(n-{(1s,3s)-3-[methyl(7h-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclobutyl}propane-1-sulfonamide hydrochloride)
(n-{(1s,3s)-3-[methyl(7h-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclobutyl}propane-1-sulfonamide hydrochloride)

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:
Test system
EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2 , Batch no.: 19-EKIN-017.
This model is a three-dimensional human epidermis model, which consists of adult human derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Application/Treatment of the Test Item
The test was performed on a total of 3 tissues per test item together with negative and positive controls. The skin was moistened with 5 µL Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue and the solid test item (17.5 to 23.2 mg) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 µL PBS (negative control) and 3 tissues with 25 µL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. Negative and positive controls were shared with parallel studies. All information pertaining to shared tissues are archived in the raw data. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.
Cell Viability Measurement
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3hat 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labeled microtubes and extracted with 500 µL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 69 hours. The amount of extracted formazan was determined spectrophotometrically at 570nmin duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
17.5 to 23.2 mg
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hours at 37°C.
Number of replicates:

Results and discussion

In vitro

Irritation / corrosion parameter:
% tissue viability
Run / experiment:
> 120
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
In conclusion, PF-04965842-01 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.
Executive summary:

The objective of this study was to evaluate PF-04965842-01 for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKINSMTM)). The possible skin irritation potential of PF-04965842-01 was tested through topical application for 15 minutes.
The study procedures described in this report were based on the most recent OECD and EC guidelines.
Batch 19-AN-00220 of PF-04965842-01 was a white solid. Skin tissue was moistened with 5 µL of Milli-Q water and at least 10 mg of PF-04965842-01 was a applied directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.
Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with PF-04965842-01compared to the negative control tissues was 120%. Since the mean relative tissue viability forPF-04965842-01 was above 50% after 15 ± 0.5 minutes treatment PF-04965842-01 is considered to be non-irritant.
The positive control had a mean cell viability of 13% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 9%, indicating that the test system functioned properly.
In conclusion, PF-04965842-01 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

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