N-[4-[4-[(dimethylamino)methyl]-3-phenyl-1H-pyrazol-1-yl]pyrimidin-2-yl]-6-methoxy-4-(morpholin-4-yl)benzene-1,3-diamine

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 2020-05-12 to 2020-05-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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:

adopted 18 June 2019

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 (incl. QA statement)

Test material

Test material information

1

Reference substance name:

N-[4-[4-[(dimethylamino)methyl]-3-phenyl-1H-pyrazol-1-yl]pyrimidin-2-yl]-6-methoxy-4-(morpholin-4-yl)benzene-1,3-diamine

Cas Number:

1903009-56-2

Molecular formula:

C27H32N8O2

IUPAC Name:

N-[4-[4-[(dimethylamino)methyl]-3-phenyl-1H-pyrazol-1-yl]pyrimidin-2-yl]-6-methoxy-4-(morpholin-4-yl)benzene-1,3-diamine

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch (Lot) No. of test material: I20AD0249
- Expiration date of the lot/batch: 2022-01-21 (retest date)
- Physical description: slightly yellow powder
- Purity test date: 2020/02/04
- Purity: 99.9% (w/w)

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
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: not indicated


OTHER SPECIFICS:
- The correction factor is not applicable for this test therefore no correction was made for the purity/composition of the test item.

In vitro test system

Test system:

human skin model

Remarks:

model of human-derived epidermal keratinocytes

Source species:

human

Cell type:

non-transformed keratinocytes

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 (EPI-200) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Tissue batch number(s): Lot no. 30865 kit W+N
- This model consists of normal human-derived 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 multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts.
- On the day of receipt the tissues were transferred to 6-well plates and pre-incubated with 0.9 mL Assay Medium for 60 ± 5 minutes at 37°C and thereafter for 20 hours at 37°C in 0.9 mL fresh pre-warmed Assay medium.

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 last 25 minutes of test item incubation at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 55 - 90%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C ( actual range 36.3 - 36.8°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 (35 ± 1.0 minutes at 37.0 ± 1.0°C and the remaining period of the 60 ± 1 minutes test item exposure at room temperature), the tissues were thoroughly rinsed with Dulbecco's phosphate buffered saline (DPBS) to remove residual test item. Cotton wool swabs were used to remove any remaining test item. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 0.9 mL pre-warmed assay medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 25 hours at 37°C. The tissues were transferred to 0.9 mL fresh Assay medium and placed back for a post-incubation period of 17.5 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 24-wells plate prefilled with 0.3 mL MTT-medium (1.0 mg/mL). The tissues were incubated for 3 hours ± 5 minutes at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. The tissues were washed with DPBS and formazan was extracted with 2 mL isopropanol for at least 2 hours at room temperature with gentle shaking. 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: The test item was checked for possible direct MTT reduction and color interference in the Skin corrosion test using EpiDerm as a skin model (Test Facility Study No. 20231712). Because solutions did not turn blue / purple and/or a blue / purple precipitate was observed it was concluded that the test item did not interfere with the MTT endpoint.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Tissue viability (OD(540-570 nm) (1.0-3.0): 1.727 +/- 0.096
- Barrier function (ET-50 ; 4.77- 8.72 hrs ): 6.5 hrs
- Contamination: sterile
- Expiration date: not indicated

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 60 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 60 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): 25.5 to 42.3 mg

NEGATIVE CONTROL (DPBS)
- Amount(s) applied (volume or weight): 30 µL

POSITIVE CONTROL (5% SDS)
- Amount(s) applied (volume or weight): 30 µL

Duration of treatment / exposure:

60 +/- 1 minutes

Duration of post-treatment incubation (if applicable):

42.5 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:

1

Value:

98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD: 4.3

Remarks:

Individual values: 102, 97 and 94

Results 2

Irritation / corrosion parameter:

other: Optical density

Run / experiment:

1

Value:

1.763

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD: 0.078

Remarks:

Individual values: 1.846, 1.750, 1.692

Other effects / acceptance of results:

mean tissue viability (percentage of control):
negative control: 100 (89, 100 and 111)
positive control: 3.5 (3.3, 3.6 and 3.5)

mean optical density:
negative control: 1.806 +/- 0.193
positive control: 0.063 +/- 0.002

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 Skin corrosion test using EpiDerm as a skin model (Test Facility Study No. 20231712). Because no color change was observed by adding MTT-medium it was concluded that the test item did not interact 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 60 ± 1 minutes treatment with the test item compared to the negative control tissues was 98% (102, 97 and 94%). 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 60 ± 1 minutes exposure of 3.5% (3.3, 3.6 and 3.5%). The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range, and within the acceptance limits of OECD439 (lower acceptance limit ≥1.0 and upper acceptance limit ≤ 2.8). The standard deviation value of the percentage viability of three tissues treated identically was ≤11%, 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 this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.