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Diss Factsheets
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EC number: 951-985-7 | 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 irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Experimental Phase: 08 July 2020 to 13 July 2020.
- 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 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)
- Deviations:
- no
- GLP compliance:
- yes
Test material
- Reference substance name:
- 2-amino-N-{6-[(2-aminophenyl)formamido]-2-{3-[(2-aminophenyl)formamido]propyl}hexyl}benzamide
- EC Number:
- 951-985-7
- Molecular formula:
- C30H38N6O3
- IUPAC Name:
- 2-amino-N-{6-[(2-aminophenyl)formamido]-2-{3-[(2-aminophenyl)formamido]propyl}hexyl}benzamide
- Test material form:
- solid: crystalline
Constituent 1
In vitro test system
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- Supplier: EpiSkin Laboratories, Lyon, France.
Date received: 07 July 2020
EpiSkinTM Tissues (0.38cm2)
Lot number: 20-EKIN-028
Maintenance Medium lot number: 20-MAIN3-018
Assay Medium lot number: 20-ESSC-018
Main Test
Application of Test Item and Rinsing (Day 1)
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. 5 µL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test item and the epidermis. Approximately 10 mg (26.3 mg/cm test item was then applied to the epidermal surface ensuring uniform covering. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls.
The plates were kept in the biological safety cabinet at room temperature for 15 minutes.
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37°C, 5% CO2 in air for 42 hours.
MTT Loading/Formazan Extraction (Day 3)
Following the 42-hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer (-35 to -10 ºC) for possible inflammatory mediator determination.
2 mL of MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells with any excess maintenance medium from the bottom of the tissue insert removed by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C,5% CO2 in air. At the end of the 3-hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Following mixing on a vortex mixer the tubes were refrigerated at 2 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.
Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution.
For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a 96-well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density (OD570) was measured (quantitative viability analysis) at 570 nm (without a reference filter) using a Labtech LT-4500 microplate reader. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- Approximately 10 mg (26.3 mg/cm) test item was then applied to the epidermal surface ensuring uniform covering.
- Duration of treatment / exposure:
- 15 minutes
- Duration of post-treatment incubation (if applicable):
- The post-exposure incubation period was 42-hour.
- Number of replicates:
- Tripilcate tissues were treated for the test item and positive and negative controls.
Results and discussion
In vitro
Results
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Test Item
- Value:
- 95.6
- 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:
- ACCEPTANCE CRITERIA
The relative mean tissue viability for the positive control treated tissues was 2.8% relative to the negative control treated tissues and the standard deviation value of the viability was 0.5%. The positive control acceptance criteria were therefore satisfied.
The mean OD570 for the negative control treated tissues was 0.845 and the standard deviation value of the viability was 9.8%. The negative control acceptance criteria were therefore satisfied.
The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 1.8%. The test item acceptance criterion was therefore satisfied.
Any other information on results incl. tables
Direct MTT Reduction
The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.
Assessment of Colour Interference with the MTT endpoint
The solution containing the test item was colourless. It was therefore unnecessary to run colour correction tissues.
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- In this study and under the experimental conditions reported, the test item was classified as non-irritant. The following classifications apply: EU CLP Not classified for Irritation. UN GHS Not classified for Irritation (category 3 cannot be determined).
- Executive summary:
Introduction
The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model. The test was designed to be compatible with the following guidelines:
- OECD Guideline for the Testing of Chemicals No. 439 In Vitro Skin Irritation: Reconstructed Human EpiDermis (RHE) Test Method (18 June 2019)
- Method B.46. in vitro skin irritation: Reconstructed Human Epidermis Model Test as described in Commission Regulation (EC) No. 640/2012, of 06 July 2012 amending, for the purpose of its adaption to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)
Method
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42-hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.
At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 570 nm.
Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).
Results
The relative mean viability of the test item treated tissues was 95.6% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.
Conclusion
In this study and under the experimental conditions reported, the test item was classified as non-irritant. The following classifications apply: EU CLP Not classified for Irritation. UN GHS Not classified for Irritation (category 3 cannot be determined).
- OECD Guideline for the Testing of Chemicals No. 439 In Vitro Skin Irritation: Reconstructed Human EpiDermis (RHE) Test Method (18 June 2019)
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