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EC number: 244-320-0 | CAS number: 21295-57-8
- 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 irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 019
- Report date:
- 2019
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
- Principles of method if other than guideline:
- This in vitro study was performed to assess the irritation potential of Macrolex Fluoreszenzrot 4B by means of the Human Skin Model Test Test
- GLP compliance:
- yes
Test material
- Reference substance name:
- 6-(cyclohexylamino)-3-methyl-3H-dibenz[f,ij]isoquinoline-2,7-dione
- EC Number:
- 244-320-0
- EC Name:
- 6-(cyclohexylamino)-3-methyl-3H-dibenz[f,ij]isoquinoline-2,7-dione
- Cas Number:
- 21295-57-8
- Molecular formula:
- C23H22N2O2
- IUPAC Name:
- 10-(cyclohexylamino)-14-methyl-14-azatetracyclo[7.7.1.0²,⁷.0¹³,¹⁷]heptadeca-1(16),2,4,6,9,11,13(17)-heptaene-8,15-dione
- Test material form:
- solid
- Details on test material:
- Purity: 94.2%
Constituent 1
In vitro test system
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Justification for test system used:
- Human full thickness skin models and reconstituted epidermal equivalents are in vitro engineered tissue cultures that provide a three dimensional architecture which is biochemically, morphologically and functionally comparable to human epidermal tissue/skin in vivo. In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and EpiSkin™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.
MatTek Corporation Protocol: In vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT); Version 11 July 2017 - Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™; Epi-200-SIT Kit and MTT-100 kit (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number(s): Lot No. 30825
- Date of initiation of testing: 30 August 2019
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: incubator (37 ± 1.5° C) and 5 ± 5% CO2 in DMEM Medium
INCUBATION TIME
main experiment: treatment time 60 min followed by a post-treatment incubation time of about 41.5 h
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Versamax® Molecular Devices
- Wavelength: 570 nm
NUMBER OF REPLICATE TISSUES:
test item and controls were tested in triplicate
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
the optical density of the isopropanol-extracts of the triplicate cultures was determined in duplicate per insert = 6 OD values.
PREDICTION MODEL / DECISION CRITERIA:
- The mean optical density (OD) value obtained with the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100 %.
- A category of UN GHS (Category 2 or Category 1) is predicted if the mean percent tissue viability after exposure and post treatment incubation is less than or equal (≤ ) to 50 %.
Pre-check for potential optical interferences of the test item
Optical properties of the test item or its chemical action on MTT may interfere with the measurement of MTT formazan leading to a false estimate of tissue viability.
The test item was therefore tested in advance for a potential direct influence on the test results not related to cytotoxic effects on tissue cells. For this pre-check the following parameters were tested:
1. Assessment of potential direct MTT-reduction of the test item
In case of a direct MTT-reduction of the test item a killed tissue control (inserts, which were killed by freezing) was used in the main assay.
2. Assessment of potential interference of colored or staining test items, which become colored after application to the tissues, with OD read out
2.1. Assessment of the color reaction with water
2.2. Assessment of the color reaction with isopropanol
In case of an influence of test item color on OD measurement, a color control was used in the main assay.
The evaluation criteria for the pre-check were:
- For MTT reduction (visual assessment): If the MTT solution color turns blue/purple, the test item is presumed to have reduced the MTT. A killed control (PG KC) must be conducted.
- For color reaction (measurement of the OD): If, after subtraction of the OD for water or isopropanol the OD of the test item solution is >0.08 a Color Control (PG CC) must be conducted.
If a test item is identified as producing both, color interference and direct MTT reduction, a third set of controls (Non-specific killed control NsKC) will be require, additionally from the PG KC and PG CC controls. This is usually the case with darkly coloured test items interfering with the MTT assay (e.g., blue, purple, black) because their intrinsic colour impedes the assessment of their capacity to directly reduce MTT.
Procedure color control
Color control (CC): The test item was applied to two additional inserts which were in principle treated as described for the main assay.
However the further processing for the determination of cell viability differs in that these color control inserts were placed in MTT-Assay medium instead of placing them in MTT solution.
The irritating potential of the test item is assessed by determination of its cytotoxic effect on an in vitro reconstructed human epidermis. The test principle is based on the MTT assay reflecting the cell viability after exposure to the topically applied test item.
All tests were performed in triplets for each time point. The test item was applied at a 100% concentration, i.e. 25 mg per insert for 60 min. Cell viability was measured by the amount of MTT reduction (calculated on the basis of optical density of the negative control). - Control samples:
- yes, concurrent vehicle
- yes, concurrent positive control
- yes, concurrent MTT non-specific colour control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied: 25 mg (39.7 mg/cm² according to guideline) wetted with 25 µL DPBS (buffer)
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μl
- Concentration (if solution): Dulbecco's Phosphate Buffered Saline (DPBS)
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl
- Concentration (if solution): 5% Sodium Dodecyl Sulfate (SDS) solution in deionised water - Duration of treatment / exposure:
- The incubation of the treated Epi-200 STI inserts was carried out for 60 min.
- Duration of post-treatment incubation (if applicable):
- about 41.5 hours
- Number of replicates:
- 3 per test item
Results and discussion
In vitro
Results
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- mean of 3 replicate cultures
- Value:
- 94.32
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Other effects / acceptance of results:
- DEMONSTRATION OF TECHNICAL PROFICIENCY:
- Reliability of the test was previously confirmed by interlaboratory validation
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: yes
- Colour interference with MTT: yes
Any other information on results incl. tables
All acceptance criteria were met.
The test item proved to be an MTT reducer in the MTT interference pre-experiment. Also, it changed colour when mixed with isopropanol. Therefore, additional tests with freeze-killed tissues, viable tissues and Non-specific Killed Control (NSKC) tissues (without MTT addition) had to be performed, see following table:
color of test item | MTT reduction | Color reaction in isopropanol | Killed control test to be conducted | Color control test to be conducted |
red | + | + | yes | yes |
+ positive reaction, - negative reaction
As can be seen from the information in the table below the test item did not show a significant impact on cell viability and is thus identified as non-irritant substance in this test model.
Summary of results:
Test item | OD mean | % Cell Viability | Evaluation of result |
Macrolex Fluoreszenzrot 4B | 1.823 | 94.32* | irritant |
negative control (NaCl 0.9%) | 1.875 | 100 | non-irritant |
positive control (SDS 5%) | 0.067 | 3.57 | non-irritant |
* Corrected mean viability = TI viability – TI_CC viability – (TI_KC viability – NC_KC viability) + TI_NSKC viability
Thus the test item does not require classification according to UN GHS (Category 2 or Category 1) under the experimental conditions described in this report.
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Executive summary:
This in vitro study was performed to assess the irritation potential of Macrolex Fluoreszenzrot 4B by means of the Human Skin Model Test (OECD TG 439).
The test item proved to be an MTT reducer in the MTT interference pre-experiment. Also, it changed colour when mixed with isopropanol. Therefore, additional tests with freeze-killed tissues, viable tissues and Non-specific Killed Control (NSKC) tissues (without MTT addition) had to be performed.
Each three tissues of the human skin model EpiDerm™ were treated with the solid test item (wetted with phosphate buffer), the negative or the positive control for 60 minutes followed by a post-treatment incubation period of 40.5 hours. The results obtained with the positive and negative controls ensured the validity of the test system
After treatment with the test item Macrolex Fluoreszenzrot 4B the mean relative viability value decreased to 94.32% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, in this study and under the experimental conditions reported, Macrolex Fluoreszenzrot 4B is non-irritant to skin according to UN GHS and EU CLP regulation.
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