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EC number: 841-500-6 | CAS number: 1903008-80-9
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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:
- 2 020
- 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
- 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
Constituent 1
- 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
Resultsopen allclose all
- 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
- 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
- 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.
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