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

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 2020-05-12

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:

18 June 2019

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

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.

Version / remarks:

20 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-[4-[(dimethylamino)methyl]-3-phenyl-1H-pyrazol-1-yl]-N-[2-methoxy-4-(morpholin-4-yl)-5-nitrophenyl]pyrimidin-2-amine

Cas Number:

1903009-55-1

Molecular formula:

C27H30N8O4

IUPAC Name:

4-[4-[(dimethylamino)methyl]-3-phenyl-1H-pyrazol-1-yl]-N-[2-methoxy-4-(morpholin-4-yl)-5-nitrophenyl]pyrimidin-2-amine

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: I19JD2758
- Expiration date of the lot/batch: 2021-10-11
- Physical Description: Yellow powder
- Purity : 99%
- Purity test date: 2019-11-28

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: The solid test item was applied directly on top of the skin tissue.

OTHER SPECIFICS: 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) obtained from MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Tissue batch number(s): 30865
- 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.
- Before the assay was started the tissues were transferred to new 6-well plates containing 0.9 mL Assay medium per well. At least 25 mg solid was added into the 6-well plates on top of the skin tissues. Tissues were moistened before application of the test item with DPBS (25 µL), to ensure close contact to the tissue. Three tissues were treated with 30 µL DPBS (negative control) and 3 tissues with 30 µL 5% SDS (positive control) respectively. Negative
and positive controls were shared with parallel studies. All information pertaining to shared tissues are archived in the raw data

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 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
- Temperature of post-treatment incubation (if applicable): 37°C
- All incubations, with the exception of the test item incubation of the last 25 minutes of exposure at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (55 - 90%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (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 with the test item (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 are 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
- 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. 20231917). 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.
- 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 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.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function (ET-50 ; 4.77- 8.72 hrs ): 6.5 hrs
- 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 the absence of bacteria, fungus and mycoplasma was verified.
- 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 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): 27.3 to 36.4 mg

NEGATIVE CONTROL (PBS)
- 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 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:

10

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD: 0.6

Remarks:

Individual values: 99, 100 and 99

Results 2

Irritation / corrosion parameter:

other: Optical density

Run / experiment:

1

Value:

1.798

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD: 0.010

Remarks:

Individual values: 1.794, 1.809, and 1.789

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. 20231917). Because no color change was observed by adding MTT-medium it was concluded that the test item did not interact with the MTT endpoint.
The mean tissue viability obtained after 60 ± 1 minutes treatment with the test item compared to the negative control tissues. 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 100% (99, 100 and 99%). 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.