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EC number: - | CAS number: -
- 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
Skin irritation / corrosion
Administrative data
- Endpoint:
- skin corrosion: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 2015-12-14 to 2016-05-27
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 016
- Report date:
- 2016
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
- Version / remarks:
- 28 July 2015
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142, 31 May 2008
- Version / remarks:
- 31 May 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- 4-[(1-[[6-cyano-5-(trifluoromethyl)pyridin-3-yl]carbamoyl]cyclobutyl)amino]-2-fluoro-N-methylbenzamide
- Cas Number:
- 1950587-20-8
- Molecular formula:
- C20H17F4N5O2
- IUPAC Name:
- 4-[(1-[[6-cyano-5-(trifluoromethyl)pyridin-3-yl]carbamoyl]cyclobutyl)amino]-2-fluoro-N-methylbenzamide
- Test material form:
- solid: particulate/powder
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Batch (Lot) No.of test material: I15DD1622
- Expiration date of the lot/batch: 2017-04-15 (retest date)
- Physical Description: Brown crystalline powder with lumps
- Purity: 103.0% (w/w) (UHPLC assay)
- Purity test date: 2015-10-15
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 (e.g. stock crystals ground to fine powder using a mortar and pestle): none, the solid test item was applied directly on top of the skin tissue.
OTHER SPECIFICS
- Correction factor: 1
- pH (1% in water, indicative range): 5.9 – 5.8 (determined by WIL Research Europe)
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:
- Recommended test system in international guidelines (OECD and EC).
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200) supplied by MatTek Corporation, Ashland MA, U.S.A
- Tissue batch number(s): 23939 kit L and M
- The 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 multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.
- On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 mL DMEM medium (Dulbecco’s Modified Eagle’s Medium) (supplemented DMEM medium, serum-free supplied by MatTek Corporation) per well. The level of the DMEM medium was just beneath the tissue. The plates were incubated for approximately 2 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM medium just before the test item was applied.
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature during 3-minute exposure and 37°C during 1-hour exposure
- Temperature of post-treatment incubation (if applicable): 37°C
- All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 62 - 87%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.7 - 36.6°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, the tissues were washed with phosphate buffered saline (PBS) (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item. Rinsed tissues were kept in 24 well plates on 300 µL DMEM medium until 6 tissues (= one application time) were dosed and rinsed.
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT medium: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.
- The DMEM medium was replaced by 300 µL MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
- Test for the interference with the MTT endpoint: A test item may interfere with the MTT endpoint if it is coloured 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.
- Test for colour interference by the test item: the test item was checked for possible colour interference before the study was started. Some non-coloured test items may change into coloured items in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the colour interference, approximately 25 mg of the test item or 50 µL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple colour change was observed.
- Test for reduction of MTT by the test item: the test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, at least 25 mg of the test item or 50 µL Milli-Q water as a negative control were added to 1 mL MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. At the end of the exposure time it was checked if a blue / purple colour change or a blue/purple precipitate was observed.
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability (OD (540-570 nm)<1.0-3.0>): 1.726 +/- 0.026
- Barrier function (ET-50 <4.77-8.72 hrs>): 6.45 hours
- Sterility: Sterile
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
DECISION CRITERIA
A test item is considered corrosive in the skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.
A test item is considered non-corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 27.5 to 29.0 mg
NEGATIVE CONTROL (Milli-Q water)
- Amount(s) applied (volume or weight): 50 µL
POSITIVE CONTROL (8N KOH)
- Amount(s) applied (volume or weight): 50 µL - Duration of treatment / exposure:
- 3 minutes and 1 hour
- Duration of post-treatment incubation (if applicable):
- 3 hours
- Number of replicates:
- 4 replicates per test item, negative control and positive control: 2 for the 3-minute exposure and 2 for the 1-hour exposure
Results and discussion
In vitro
Resultsopen allclose all
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Experiment 2: 3-minute application
- Value:
- 103
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Individual values: 100 and 106
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Experiment 2: 1-hour application
- Value:
- 97
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Individual values: 96 and 99
- Irritation / corrosion parameter:
- other: optical denisty
- Run / experiment:
- Experiment 2: 3-minute application
- Value:
- 1.79
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: SD +/- 0.075; Individual values: 1.737, 1.844
- Irritation / corrosion parameter:
- other: optical density
- Run / experiment:
- Experiment 2: 1-hour application
- Value:
- 1.69
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: SD +/- 0.040; Individual values: 1.662, 1.718
- Irritation / corrosion parameter:
- other: Maximum inter-tissue variability in viability between two tissues treated identically
- Run / experiment:
- 3-minute application
- Value:
- 5.8
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Maximum difference in percentage between the mean viability of two tissues and one of the two tissues: 3.0 and 2.9
- Irritation / corrosion parameter:
- other: Maximum inter-tissue variability in viability between two tissues treated identically
- Run / experiment:
- 1-hour application
- Value:
- 3.3
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Maximum difference in percentage between the mean viability of two tissues and one of the two tissues: 1.7 and 1.6
- Other effects / acceptance of results:
- 3-minute application:
- viability (percentage of control) (range):
negative control: 100 (102 and 98)
positive control: 13 (16 and 10)
- Maximum inter-tissue variability in viability between two tissues treated identically:
negative control: 3.8
positive control: 37.7
- Maximum difference in percentage between the mean viability of two tissues and one of the two tissues:
negative control: 1.9 and 1.9
positive control: 18.9 and 23.3
- mean optical density:
negative control: 1.739 +/- 0.048
positive control: 0.223 +/- 0.073
1-hour application:
- viability (percentage of control) (range):
negative control: 100 (104 and 96)
positive control: 7 (8 and 6)
- Maximum inter-tissue variability in viability between two tissues treated identically:
negative control: 6.9
positive control: 20.0
- Maximum difference in percentage between the mean viability of two tissues and one of the two tissues:
negative control: 3.5 and 3.6
positive control: 10.0 and 11.1
- mean optical density:
negative control: 1.737 +/- 0.088
positive control: 0.124 +/- 0.020
The test item was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that the test item did not interfere with the MTT endpoint.
Since for the first test no certificate of analysis of the skin tissues was available due to a barrier function above the limits of the supplier this test was rejected and a repeat test was performed. The results of both tests were comparable.
Results:
Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 103% (100 and 106%) and 97% (96 and 99%) respectively. Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item is considered to be not corrosive.
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3-minute exposure to the positive control was 13% (16 and 10%) and 7% (8 and 6%) after 1 hour exposure. The maximum inter-tissue variability in viability between two tissues treated identically was less than 7% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 4% for the negative control and test item. For the positive control, the maximum inter-tissue variability in viability between two tissues treated identically was less than 38% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 24%, however since the viabilities were below 20% the acceptability criteria were met. It was therefore concluded that the test system functioned properly.
Any other information on results incl. tables
Results on rejected experiment:
3-minute application:
- viability (percentage of control) (range):
negative control: 100 (111 and 89)
positive control: 22 (17 and 28)
test item: 90 (93 andf 86)
- Maximum inter-tissue variability in viability between two tissues treated identically:
negative control: 19.8
positive control: 41.5
test item: 7.5
- Maximum difference in percentage between the mean viability of two tissues and one of the two tissues:
negative control: 9.9 and 11.0
positive control: 26.2 and 20.8
test item: 3.7 and 3.9
- mean optical density:
negative control: 1.983 +/- 0.307
positive control: 0.444 +/- 0.165
test item: 1.784 +/- 0.098
1-hour application:
- viability (percentage of control) (range):
negative control: 100 (105 and 95)
positive control: 7 (4 and 10)
test item: 82 (89 and 75)
- Maximum inter-tissue variability in viability between two tissues treated identically:
negative control: 9.7
positive control: 53.2
test item: 14.8
- Maximum difference in percentage between the mean viability of two tissues and one of the two tissues:
negative control: 4.8 and 5.1
positive control: 36.3 and 26.6
test item: 7.4 and 8.0
- mean optical density:
negative control: 2.238 +/- 0.161
positive control: 0.157 +/- 0.080
test item: 1.835 +/- 0.208
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- It is concluded that the test is valid and that the test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in the report.
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