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EC number: 812-745-6 | CAS number: 205041-15-2
- 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:
- 2020-04-22 to 2020-08-14
- 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 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
- Version / remarks:
- adopted in June 2018
- Deviations:
- no
- GLP compliance:
- yes
- Type of study:
- activation of keratinocytes
- Justification for non-LLNA method:
- 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 skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD 442D).
Test material
- Reference substance name:
- (prop-2-en-1-yl)({3-[(prop-2-en-1-yl)amino]propyl})amine dihydrochloride
- EC Number:
- 812-745-6
- Cas Number:
- 205041-15-2
- Molecular formula:
- C9 H20 Cl2 N2
- IUPAC Name:
- (prop-2-en-1-yl)({3-[(prop-2-en-1-yl)amino]propyl})amine dihydrochloride
- Test material form:
- solid
- Details on test material:
- - CAS: 205041-15-2
- Batch: LWDDP00087
- Colour: white
- Purity (GC): 99%
- Expiry date: 2020-09-25
Constituent 1
- Specific details on test material used for the study:
- TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of the experiment (immediately before treatment) N1,N3-diallylpropane-1,3-diamine dihydrochloride was dissolved or stably dispersed/suspended in DMSO to prepare a stock solution with a concentration of 2064 mM. Due to a technical error in the pre-experiment, the top dose was higher than required by the OECD Guideline 442D.
OTHER SPECIFICS
Partition coefficient (n-octanol/water): -4.78
In vitro test system
- Details on the study design:
- Test System:
- The LuSens cell-line is an immortalised adherent cell line derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The plasmid contains a luciferase gene (reporter gene) which is under transcriptional control of an antioxidant response element (ARE) of the rat NQO1 gene.The HaCaT human keratinocytes (provided by RWTH, Aachen, Germany) were genetically modified at the Institute of Anatomy and Cell Biology of the RWTH, Aachen (laboratory of Wruck). The LuSens cells were obtained by BASF and in 2019 a contract services agreement was established between ICCR-Roßdorf GmbH (Licensee) and Promega.
Cell Culture:
- Cultivation medium : Dulbecco´s Modified Eagle Medium (DMEM) culture medium with Penicillin/Streptomycin (1% v/v), FBS (10% v/v) and puromycin (0.005% v/v).
- Seeding medium: DMEM culture medium with FBS (10% v/v)
- Treatment medium: DMEM culture medium with FBS (1% v/v)
- LuSens Cell Cultures: Stocks of the LuSens cell line were stored in liquid nitrogen in the cell bank of ICCR-Roßdorf GmbH (aliquots of cells in freezing medium at 1.5 - 5×10^6 cells/cryovial) allowing the repeated use of the same cell culture batch in experiments. Therefore, the parameters of the experiments remain similar, because of the reproducible characteristics of the cells. The stock cultures were thawed at 37 ± 1.5 °C in a water bath. The cells were sub-cultured twice weekly. The cell density should not exceed a cell density of 80 – 90%. The confluent cells were incubated at 37 ± 1.5 °C and 5.0 ± 0.5 % CO2 (standard cell culture conditions). The LuSens cells can be used up to one and a half month after thawing (passage number should not exceed passage 15 for the main experiment and passage 20 for the dose range finder).The passage numbers of the used LuSens cells were 5 in the cytotoxicity test and 9/K1 and 9/K2 in the LuSens test for the main experiments 3 and 4, respectively.
Preparation and seeding of the cells:
Between 4 and 6×10^5 or 6 and 8×10^5 cells were seeded in 15 mL Cultivation Medium and pre-cultured at least twice a week in culture flasks (80 – 90% confluent). The cell density between approximately 80 and 90% should not be exceeded. After microscopic assessment of the cells, the cells were washed at least twice with 10 mL Ca2+/Mg2+ free DPBS including EDTA. Thereafter, the cells were trypsinated with 1 to 2 mL 0.05% Trypsin/EDTA for approximately 5 minutes at 37 ± 1.5 °C and 5.0 ± 0.5% CO2. The cells were resuspended in 10 mL Cultivation Medium to neutralise the trypsin.
For seeding of the cells, the Cultivation Medium was removed, and the cells were transferred into Seeding Medium. Each treatment well of a 96 well microtiter plate was seeded with 100 μL cell suspension (9000 to 11000 LuSens cells per well), except the well of the blank control. The cells were incubated for 24 hours ± 30 minutes under standard cell culture conditions.
Main Experiment (LuSens and MTT):
The test item was tested in four independent main experiments. The first two experiments were canceled due to observed contaminations. The main experiments were performed with independent cell cultures (cells are collected from different culture flasks). The test item was prepared separately for each run. For each main experiment one 96 well microtiter plate was prepared for MTT assay and one for the luciferase activity measurement.
Treatment of the Cells:
The solvent (twenty-four replicates), positive (five replicates) and negative (six replicates) controls as well as the test item concentrations (three replicates for each concentration) were diluted 1:25 in Treatment Medium. After incubation of the LuSens cells, Seeding Medium was removed and 150 µL of Treatment Medium was distributed in each well. Thereafter, 50 µL of the test item and control dilutions and the medium control (twelve replicates) were added into the corresponding wells. At the end of
the incubation period of 48 ± 1 hours under standard cell culture conditions, the cell cultures were microscopically evaluated for morphological alterations, precipitation or phase separation.
MTT assay (cell viability):
At the end of the incubation period, Treatment Medium was removed from the wells and the cells were washed at least twice with 200 µL DPBS including Ca2+/Mg2+. Thereafter, 200 µL of the MTT working solution were added to each treatment well and the cells were incubated for 3 hours ± 30 minutes under standard cell culture conditions. After rinsing the MTT working solution, the cells of each well were treated with 100 µL MTT lysis agent (Isopropanol with 0.04 N HCl) for at least 30 minutes, while gently shaking. Thereafter the microplate was transferred to a microplate reader equipped with a 570 nm filter to measure the absorbance (reference wavelength 690 nm).
Luciferase activity measurement:
- The Steady-Glo® Mix was be prepared by adding Steady-Glo® buffer to one bottle of Steady-Glo® Substrate and mixing by inversion until the substrate was dissolved. The Steady-Glo® working solution was prepared by mixing one part of DPBS (without Ca^2+/Mg^2+) with one part of Steady-Glo®-Mix.
At the end of the incubation period, the Treatment Medium was removed from the wells and the cells were washed at least twice with 200 μL DPBS including Ca^2+/Mg^2+. Thereafter, 200 μL of the Steady-Glo® working solution was added in each well. After slowly shaking of the microtiter plate for at least 10 min in the dark, the plate was transferred to a microplate reader and the luminescence was measured for 2 seconds per well.
Dose groups:
- Medium control: Treatment medium
- Solvent control: DMSO (final concentration 1% (v/v) in Treatment Medium), purity: ≥ 99%
- Negative control: Lactic acid (final concentration 5000 μM), CAS 50-21-5, purity: ~ 90%
- Positive Control: Ethylene glycol dimethylacrylate (EGDMA) (final concentration 120 μM), CAS 97-90-5, purity: ≥ 97.5%
- Test Item, main experiments: 178, 214, 257, 308, 370, 444 μM
Results and discussion
- Positive control results:
- The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥ 2.5 (main experiment (ME) 3: 6.878; ME 4: 5.603) and statistically significant. The positive control also had a relative cell viability ≥ 70% as compared to the solvent control (ME 3: 88.87%; ME 4: 114.00%).
In vitro / in chemico
Resultsopen allclose all
- Key result
- Run / experiment:
- other: Experiment 3
- Parameter:
- other: highest luciferase induction with a cell viability > 70% (at 178 µM)
- Value:
- 1.466
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Run / experiment:
- other: Experiment 4
- Parameter:
- other: highest luciferase induction with a cell viability > 70% (at 370 µM)
- Value:
- 1.4
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Other effects / acceptance of results:
- The acceptance criteria were met:
- The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥ 2.5 (ME 3: 6.878; ME 4: 5.603) and statistically significant.
- The positive control had a relative cell viability ≥ 70% as compared to the solvent control (ME 3: 88.87%; ME 4: 114.00%).
- The average luciferase activity induction obtained with the negative control, 5000 μM Lactic acid, as well as the basal expression of untreated cells was < 1.5 fold as compared to the average solvent control (ME 3: 0.872; ME 4: 0.744).
- The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) should be below 20% in each main experiment (ME 3: 13.0%; ME 4: 8.8%).
- At least three test concentrations had a cell viability of at least 70% relative to the solvent controls. At least one concentration was cytotoxic, i.e. had a cell viability < 70%.
Any other information on results incl. tables
Table 2: Luminescence induction and cell viability of the test item in Experiment 3
Treatment Group |
Concentration |
Luminescence |
Mean Luminescence |
SD Luminescence |
Mean Luminescence blank corr. |
Fold Induction |
Cell viability [%] |
|||||||||
Well 1 |
Well 2 |
Well 3 |
Well 4 |
Well 5 |
Well 6 |
|||||||||||
Blank |
|
|
|
185 |
|
|
|
|
|
|
|
|
|
|
||
Solvent control |
|
|
|
|
|
|
|
|
282.3 |
36.8 |
97.3 |
1.00 |
100.0 |
|||
Medium control |
|
|
|
|
|
|
|
|
249.9 |
19.4 |
64.9 |
0.667 |
81.66 |
|||
Positive Control |
|
120 |
µM |
1057 |
724 |
1256 |
650 |
584 |
|
854.2 |
289.1 |
669.2 |
6.878 |
88.87 |
||
Negative Control |
|
5000 |
µM |
266 |
259 |
266 |
325 |
244 |
259 |
269.8 |
28.2 |
84.8 |
0.872 |
79.73 |
||
Test Item |
C1 |
178 |
µM |
429 |
281 |
273 |
|
|
|
327.7 |
87.8 |
142.7 |
1.466 |
90.14 |
||
C2 |
214 |
µM |
318 |
296 |
251 |
|
|
|
288.3 |
34.2 |
103.3 |
1.062 |
71.70 |
|||
C3 |
257 |
µM |
340 |
244 |
229 |
|
|
|
271.0 |
60.2 |
86.0 |
0.884 |
74.50 |
|||
C4 |
308 |
µM |
303 |
281 |
259 |
|
|
|
281.0 |
22.0 |
96.0 |
0.987 |
77.03 |
|||
C5 |
370 |
µM |
288 |
273 |
251 |
|
|
|
270.7 |
18.6 |
85.7 |
0.881 |
62.35 |
|||
C6 |
444 |
µM |
288 |
281 |
281 |
|
|
|
283.3 |
4.0 |
98.3 |
1.011 |
42.61 |
Table 3: Luminescence induction and cell viability of the test item in Experiment 4
Treatment Group |
Concentration |
Luminescence |
Mean Luminescence |
SD Luminescence |
Mean Luminescence blank corr. |
Fold Induction |
Cell viability [%] |
|||||||||
Well 1 |
Well 2 |
Well 3 |
Well 4 |
Well 5 |
Well 6 |
|||||||||||
Blank |
|
|
|
207 |
|
|
|
|
|
|
|
|
|
|
||
Solvent control |
|
|
|
|
|
|
|
|
282.7 |
24.8 |
75.7 |
1.00 |
100.0 |
|||
Medium control |
|
|
|
|
|
|
|
|
290.0 |
19.0 |
83.0 |
1.096 |
123.35 |
|||
Positive Control |
|
120 |
µM |
636 |
643 |
591 |
665 |
621 |
|
631.2 |
27.5 |
424.2 |
5.603 |
114.00 |
||
Negative Control |
|
5000 |
µM |
266 |
273 |
244 |
273 |
273 |
251 |
263.3 |
12.8 |
56.3 |
0.744 |
114.25 |
||
Test Item |
C1 |
178 |
µM |
340 |
273 |
259 |
|
|
|
290.7 |
43.3 |
83.7 |
1.105 |
92.32 |
||
C2 |
214 |
µM |
281 |
273 |
236 |
|
|
|
263.3 |
24.0 |
56.3 |
0.744 |
96.44 |
|||
C3 |
257 |
µM |
273 |
340 |
266 |
|
|
|
293.0 |
40.9 |
86.0 |
1.136 |
80.83 |
|||
C4 |
308 |
µM |
281 |
251 |
259 |
|
|
|
263.7 |
15.5 |
56.7 |
0.748 |
93.42 |
|||
C5 |
370 |
µM |
325 |
318 |
296 |
|
|
|
313.0 |
15.1 |
106.0 |
1.400 |
84.66 |
|||
C6 |
444 |
µM |
362 |
355 |
281 |
|
|
|
332.7 |
44.9 |
125.7 |
1.660 |
57.79 |
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- In conclusion, the test item N1,N3-diallylpropane-1,3-diamine dihydrochloride did not activate the LuSens cells up to a concentration of 444 µM under the test conditions of this study. Therefore, the test item is considered negative for the second key event of the skin sensitisation Adverse Outcome Pathway (AOP).
- Executive summary:
- In an in vitro skin sensitisation study conducted according to OECD 442D with N1,N3-diallylpropane-1,3-diamine dihydrochloride (99% purity) in DMSO, the sensitisation potential of the test item was assessed in the LuSens keratinocyte cell line as changes in the expression of genes associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways (second key event of a skin sensitization AOP). The cells were incubated with the test item for 48 h at concentrations of up to 444 µM in two independent experiments. All acceptance criteria were met showing the validity of the study. After treatment with the test item, the luciferase induction was not above or equal to (≥) 1.5-fold compared to the solvent control in at least 2 consecutive non-cytotoxic tested concentrations. Therefore, the test item can be considered negative for the second key event of the skin sensitisation Adverse Outcome Pathway (AOP).
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