2-Propenamide, N-[5-[[4-[4-[(dimethylamino)methyl]-3-phenyl-1H-pyrazol-1-yl]-2-pyrimidinyl]amino]-4-methoxy-2-(4-morpholinyl)phenyl]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2020-10-06 to 2020-10-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

29 June 2020

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

20 July 2012

Deviations:

no

GLP compliance:

yes

Remarks:

certificate not included

Test material

Test material information

Constituent 1

Reference substance name:

N-{5-[(4-{4-[(dimethylamino)methyl]-3-phenyl-1H-pyrazol-1-yl}pyrimidin-2-yl)amino]-4-methoxy-2-(morpholin-4-yl)phenyl}prop-2-enamide

EC Number:

841-500-6

Cas Number:

1903008-80-9

Molecular formula:

C30H34N8O3

IUPAC Name:

N-{5-[(4-{4-[(dimethylamino)methyl]-3-phenyl-1H-pyrazol-1-yl}pyrimidin-2-yl)amino]-4-methoxy-2-(morpholin-4-yl)phenyl}prop-2-enamide

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot/batch number of test material: M20BD0542
- Expiry date: 2022-02-19 (retest date)
- Physical Description: Slightly greenish yellow powder
- Purity: 99.8%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under storage conditions: not indicated
- Stability under test conditions: not indicated

OTHER SPECIFICS
- Correction factor: 1.00

In vitro test system

Test system:

human skin model

Remarks:

model of human-derived epidermal keratinocytes

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult human derived epidermal keratinocytes (SkinEthic Laboratories, Lyon, France)

Source strain:

other: not applicable

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise 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).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm²), SkinEthic Laboratories, Lyon, France
- Tissue batch number(s): 20 EKIN 041
- 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.
- On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for approximately 24 hours at 37°C.
- 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 was added into 12-well plates on top of the skin tissues.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C
- All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 84 - 94%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.7 - 37.3°C).
- Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline (PBS) 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.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL solution (0.3 mg/mL in PBS). The tissues were incubated for 3 h at 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 prelabeled microtubes and extracted with 500 µL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for approximately 69 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
- Test for reduction of MTT by test item: A test item may interfere with the MTT endpoint if it is colored and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed. The test item has been tested previously for possible direct MTT reduction and color interference in the EpiOcular test using EpiOcularTM tissue as a model (Test Facility Study No. 20264647).

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function (IC50 determination, ≥ 1.5 mg/mL and ≤ 3.0 mg/mL): 2.7 mg/ml
- Morphology (number of cell layers ≥4): multi layered, highly differentiated epidermis consisting of organised basal, spinous and granular layers and a multilayered stratum corneum, 7 cell layers
- Contamination: on blood of the same donors, the absence of HIV1 and 2 antibodies, hepatitis C antibodies, hepatitis B antigen HBs was verified; - on cells of the same donors, the absence of bacteria, fungus and mycoplasma was verified.
- Expiration date: 2020-10-12

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

INTERPRETATION
- A test item is considered to be irritant in the skin irritation test (category 2) if: the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- A test item is considered to be non-irritant in the in vitro skin irritation test (no category) if the relative mean tissue viability after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10.8 to 20.3 mg

NEGATIVE CONTROL (PBS)
- Amount(s) applied (volume or weight): 25 µL

POSITIVE CONTROL (5% SDS)
- Amount(s) applied (volume or weight): 25 µL

Duration of treatment / exposure:

15 +/- 0.5 minutes (the positive control was re-spread after 7 minutes contact time)

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 replicates per test item together with negative and positive controls

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

experiment 1

Value:

96

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD +/- 8.0%; individual values: 88, 97 and 103%

Results 2

Irritation / corrosion parameter:

other: Optical density

Run / experiment:

experiment 1

Value:

1.122

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD +/- 0.093; individual values: 1.022, 1.138 and 1.207

Other effects / acceptance of results:

mean tissue viability (percentage of control):
negative control: 100% (99, 106 and 96%), SD: 5.1%
positive control: 8.8 % (7.5, 8.7 and 10%), SD: 1.4%

mean optical density:
negative control: 1.168 +/- 0.060
positive control: 0.103 +/- 0.017

Interpretation:
The viabilities of all replicates were within one category.

Results:
- The test item was checked for possible direct MTT reduction and color interference in the EpiOcular test using EpiOcular TM tissue as a model (Test Facility Study No. 20264647). Because the solutions did not turn blue / purple, nor a blue / purple precipitate was observed and the OD for the test item solution was ≤ 0.08, it was concluded that the test item did not interfere with the MTT endpoint.

- 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 the test item compared to the negative control tissues was 96% (88, 97 and 103%). Since the mean relative tissue viability for the test item was above 50% the test item is considered to be non-irritant. The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 8.8% (7.5, 8.7 and 10%). The absolute mean OD570 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 ≤ 8.0%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, the test item is non-irritant in the in vitro skin irritation test under the
experimental conditions described in the report and should not be classified according to the
Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the
United Nations.