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

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-09-08 to 2020-09-12

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes

Remarks:

certificate non 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

Test animals / tissue source

Species:

human

Strain:

other: normal human keratinocytes, strain 4F1188 (MatTek Corporation, Ashland MA, U.S.A.)

Details on test animals or tissues and environmental conditions:

- Justification of the test method (e.g. ICE, EIT, RhCE) and considerations regarding applicability: 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 eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications (e.g. OECD).
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from
normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture
- RhCE tissue or hCE cell construct used, including batch number: EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 31755, kit A

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 61.4 to 65.8 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 µL

Duration of treatment / exposure:

18 hours ± 15 minutes

Duration of post- treatment incubation (in vitro):

180 ± 10 minutes

Number of animals or in vitro replicates:

The test was performed on a total of 2 tissues per test item together with a negative control and positive control.

Details on study design:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiOcular™ (OCL-200-EIT MatTek Corporation)
- Tissue batch number(s): 31755
- Production date: 2020-09-09
- Shipping date: not indicated
- Delivery date: not indicated
- Date of initiation of testing: 2020-09-08
- The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm²) prepared from normal human keratinocytes (MatTek).It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells .A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ± 1.0°C
- Temperature during incubation (Post-Soak): room temperature
- All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 -100% (actual range 72-95%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0±1.0°C (actual range 35.8-37.3°C).
- Temperature and humidity were continuously monitored throughout the experiment. The CO2percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2percentage 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, the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item. After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25 ± 2 minute immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 18 hours ±15 minutes at 37°C.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (5 mg/mL) diluted (1:5)) with MTT diluent
- After incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 mL MTT-medium (1.0 mg/mL).The tissues were incubated for 180 ± 10 minutes at 37°C. After incubation with MTT-medium the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labeled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol is flowing into the insert. Formazan was extracted with 2 mL isopropanol for 2 hours at room temperature with gentle shaking.
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader.
- Wavelength: 570 nm
-Test for color interference: to assess the color interference, approximately 50 mg of the test item or 50 μLsterile Milli-Q water as a negative control was added to 1.0 mL Milli-Q water. The mixture was incubated for at least 1 hour at 37.0 ± 1.0°Cin the dark. After incubation approximately 1 mL of the mixture was centrifuged for 30 seconds at 16000 g. Furthermore, approximately 50 mg of the test item or 50 μLsterile Milli-Q water as a negative control was added to 2.0 mL isopropanol. The mixture was incubated for 2 -3 hours at room temperature with gentle shaking. After incubation approximately 1 mL of the mixture was centrifuged for 30 seconds at 16000 g. At the end of the exposure time, the absorbance of the solutions was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite®M200 Pro Plate Reader. If after subtraction of the negative control, the OD for the test item solution is >0.08, the test item is considered as possibly interacting with the MTT measurement.
- Test for reduction of MTT by the test item: to assess the ability of the test item to reduce MTT, approximately 50 mg of the test item was added to 1 mL MTT solution (1 mg/mL MTT in phosphate buffered saline). The mixture was incubated for approximately 3 hours at 37.0 ± 1.0°Cin the dark. A negative control, 50 μLsterile Milli-Q water was tested concurrently. If the MTT solution color turned blue / purple or if a blue / purple precipitate was observed the test item interacts with MTT. Only test items which bind to the tissue after rinsing can interact with MTT in the main assay.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability (OD (540-570 nm)<1.1-3.0>): 1.746 +/- 0.022
- Barrier function (ET-50 <12.2-37.5 min>): 13.59 min
- Sterility: sterile

NUMBER OF REPLICATE TISSUES: The test was performed on a total of 2 tissues per test item together with a negative control and positive control.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%. In this case no further testing in other test methods is required.
- The test chemical is identified as “no prediction can be made” if the mean percent tissue viability after exposure and postexposure incubation is less than or equal (≤) to 60%.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: difference between % tissue viabilities

Run / experiment:

1

Value:

1.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

other: optical density

Run / experiment:

1

Value:

1.683

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD +/- 0.022; individual values: 1.699 and 1.667

Results 3

Irritation parameter:

mean percent tissue viability 

Run / experiment:

1

Value:

102

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

individual values: 103% and 101%

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- mean viability (percentage of control) (individual results):
negative control: 100 (99 and 101)
positive control: 9.8 (8.9 and 11)
- coefficient of variation between tissue replicates
negative control: 1.7
positive control: 1.9
- mean optical density:
negative control: 1.645 +/- 0.019
positive control: 0.161 +/- 0.022

INTERFERENCE OF THE TEST ITEM WITH THE MTT ENDPOINT
- The test item was checked for possible direct MTT reduction by adding the test item to MTT medium. Because no color changes were observed it was concluded that the test item did not interact with the MTT endpoint. The test item was checked for color interference in aqueous conditions. Addition of the test item to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of 0.0007 and 0.0016, respectively. Therefore it was concluded that the test item did not induce color interference. In addition, because no color change was observed in the presence of MTT it was concluded that the test item did not interact with the MTT endpoint.

INTERPRETATION
- Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 6 hours ±15 minutes treatment with the test item compared to the negative control tissues was 102%(103% and 101% respectively).Since the mean relative tissue viability for the test item was above 60% the test item is considered to be non-irritant.
- The positive control had a mean cell viability after 6 hours ±15 minutes exposure of 9.8% (8.9% and 11% respectively).The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The difference between the percentage of viability of two tissues treated identically was less than 2%, indicating that the test system functioned properly.

Any other information on results incl. tables

Calculation of Cell Viability
Optical Density readings were transferred into Microsoft Excel to allow further calculations to be performed.
The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw).
ODc = ODraw – ODbl
The OD value representing 100% cell viability is the average OD of the negative controls (ODlt_u+MTT).
The %Viability for each sample and positive control is calculated as follows:
%Viability = (ODc/mean ODlt_u+MTT) * 100

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, the test item is non-irritant in the EpiOcular™ test under the experimental conditions described in the report.