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EC number: 275-276-0 | CAS number: 71216-01-8
- 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 sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2014
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP-compliant and in-house validated study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 014
- Report date:
- 2014
Materials and methods
Test guideline
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: OECD TG no. 442D (adopted February 2015) - In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method
- Principles of method if other than guideline:
- Identification of keratinocyte activating substances with the transgenic keratinocyte cell line Lu-Sens derived from HaCaT cells. lt employs the reporter gene for luciferase under the control of an antioxidant response element and hence monitors Nrf-2 transcription factor activity. and is a "me-too" assay of the KeratinoSensTM assay.
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- other: in-vitro keratinocyte activation assay (LuSens)
Test material
- Reference substance name:
- N,N''-naphthalene-1,5-diylbis[N'-[3-[(2-ethylhexyl)oxy]propyl]urea]
- EC Number:
- 275-276-0
- EC Name:
- N,N''-naphthalene-1,5-diylbis[N'-[3-[(2-ethylhexyl)oxy]propyl]urea]
- Cas Number:
- 71216-01-8
- Molecular formula:
- C34H56N4O4
- IUPAC Name:
- N',N'''-naphthalene-1,5-diylbis(1-{3-[(2-ethylhexyl)oxy]propyl}urea)
- Details on test material:
- - Physical state: solid, white
-purity: 99.5%
- Purity test date: 2014
- Lot/batch No.: 130003P040
- Expiration date of the lot/batch: unlimited stability (lot manufactured in 2013)
- Stability under test conditions: stable
- Storage condition of test material: at room temperature
- Other: The test substance appeared to be homogeneous.
Constituent 1
In vivo test system
Test animals
- Species:
- other: LuSens cells (derived from human keratinocyte cell line HaCaT)
- Details on test animals and environmental conditions:
- The reporter gene cell line LuSens was prepared in collaboration with the RWTH Aachen University. This keratinocyte cell line derived from HaCaT cells carries a reporter gene for luciferase under the control of an antioxidant-response-element (ARE) and hence monitors Nrf2 transcription factor activity. The sequence of the ARE promoter originates from the NADPH:quinone oxidoreductase1 gene from rats.
LuSens cells are routinely cultured in complete DMEM culture medium with high glucose supplemented with 10% fetal bovine serum (FBS),100 U/mL penicillin – 100 ug/mL streptomycin and 0.5 ug/mL puromycin in T75 culture flasks.
Study design: in vivo (non-LLNA)
Induction
- Concentration / amount:
- Stock solutions of the substances were prepared by dissolving in distilled water or in DMSO (final concentration of 200 mM) and diluted in 2-fold dilutions. Test substance solutions were further diluted in medium containing 1% FBS w/o puromycin to obtain a DMSO concentration of 1%.
The highest tested concentration in the main experiment was 1.22 fold of the concentration affording a viability of 75% (CV75). The additional concentrations were obtained by a 1:1exp2 dilution series of the CV75.
Cytotoxicity was determined via the MTT assay.
For substances dissolved in water, the final DMSO concentration was adjusted to 1%.
Challenge
- Concentration / amount:
- Stock solutions of the substances were prepared by dissolving in distilled water or in DMSO (final concentration of 200 mM) and diluted in 2-fold dilutions. Test substance solutions were further diluted in medium containing 1% FBS w/o puromycin to obtain a DMSO concentration of 1%.
The highest tested concentration in the main experiment was 1.22 fold of the concentration affording a viability of 75% (CV75). The additional concentrations were obtained by a 1:1exp2 dilution series of the CV75.
Cytotoxicity was determined via the MTT assay.
For substances dissolved in water, the final DMSO concentration was adjusted to 1%.
- No. of animals per dose:
- Two independent experiments were performed. In each experiment, three duplicates of each treatment were tested.
- Details on study design:
- LuSens cells from the working cell bank were thawed and cultured using culture containing antibiotics, under standard culture conditions for at least 2 weeks at passage >5 but not longer than 15 passages prior to testing.
Prior to substance incubation, cells were seeded in 96-well microtiter plates (0.12 mL of 0.83 x 10exp5 cells/ml cell suspensions), using culture medium without antibiotics for incubation for 24 hours.
Treatment was initiated by replacing regular cell culture medium with medium containing the test substance and a reduced content of FBS (1%).
Substance incubation was performed under standard cell culture conditions for 48 h and luciferase activity then determined using SteadyGlo™ (Promega, Germany) according to manufacturer’s instructions. - Positive control substance(s):
- yes
- Remarks:
- Ethylene glycol dimethacrylate (0.015 mg/ml)
Results and discussion
Any other information on results incl. tables
Concentration (test substance) µg/mL |
1st experiment fold induction rel. viability [%] |
2nd experiment fold induction rel. viability [%] |
3rd experiment fold induction rel. viability [%] |
|||||||||
mean | SD | mean | SD | mean | SD | mean | SD | mean | SD | mean | SD | |
279 (Su) | 1.08 | 0.12 | 82.9 | 3.2 | 1.10 | 0.19 | 87.6 | 7.0 | 0.96 | 0.24 | 76.7 | 5.2 |
335 (Su) | 0.95 | 0.30 | 73.9 | 3.2 | 1.03 | 0.19 | 79.7 | 4.1 | 1.18 | 0.16 | 80.3 | 3.3 |
402 (Su) | 1.01 | 0.46 | 72.1 | 6.6 | 0.94 | 0.36 | 80.9 | 5.7 | 0.99 | 0.23 | 77.7 | 4.9 |
482 (Su) | 0.89 | 0.02 | 72.9 | 2.9 | 1.08 | 0.43 | 81.0 | 1.7 | 1.17 | 0.07 | 76.6 | 2.0 |
579 (Su) | 1.67 | 0.25 | 75.3 | 3.7 | 1.30 | 0.31 | 76.6 | 3.4 | 0.99 | 0.43 | 75.2 | 8.1 |
694 (Su) | 1.22 | 0.49 | 77.8 | 7.3 | 1.16 | 0.20 | 80.6 | 3.8 | 1.04 | 0.32 | 77.0 | 9.4 |
833 (Su) | 1.86 | 0.39 | 71.3 | 6.5 | 0.83 | 0.15 | 79.5 | 2.9 | 1.20 | 0.04 | 74.4 | 2.4 |
1000 (Su) | 1.20 | 0.13 | 84.6 | 0.6 | 1.03 | 0.45 | 83.1 | 0.6 | 1.20 | 0.15 | 80.1 | 5.8 |
VC | 1.00 | 0.23 | 100.0 | 4.0 | 1.00 | 0.40 | 100.0 | 2.6 | 1.00 | 0.18 | 100.0 | 5.9 |
EGDMA (18 µg/mL) | 5.39 | 1.14 | 100.8 | 5.3 | 6.52 | 1.12 | 103.8 | 2.6 | 5.30 | 0.44 | 107.0 | 1.4 |
LA (450 µg/mL) | 1.24 | 0.21 | 116.7 | 1.5 | 1.23 | 0.32 | 119.2 | 2.4 | 1.04 | 0.27 | 113.1 | 2.8 |
Applicant's summary and conclusion
- Interpretation of results:
- other: no induction of antioxidant response genes in a keratinocyte cell line
- Executive summary:
The keratinocyte activating potential of test substance was evaluated in the LuSens assay. For this purpose the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer. In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 9 concentrations of the test substance and cytotoxicity was determined thereafter by MTT assay. No decrease in cell viability below 75% was observed up to the maximum applicable concentration of 1000 μg/mL.
In the main test luciferase activity was measured after 48 hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 3 valid experiments were performed.
The test substance was a suspension in culture medium (2 x stock solutions) and in finally tested concentrations containing 1% DMSO. After 48 hours suspensions were noticed in all concentrations. after 48 hours of exposure to test substance luciferase activity in LuSens cells was not induced affording at least 70% viability in at least two consecutive concentrations of two independent experiments. From this it has to be concluded that test substance does not have a keratinocyte activating potential up to the maximum applicable concentration of 1000 μg/mL in the LuSens under the test conditions chosen.
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