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EC number: 701-329-9 | 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 sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 01 Aug - 06 Sep 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 guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
- Version / remarks:
- adopted June 2018
- Deviations:
- yes
- Remarks:
- dose formulations were no solutions, therefore sterile filtration of dose formualtions before application was not possible
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Secrétariat général du GIPC-DGE-SI, Ivry-sur-Seine CEDEX, France
- Type of study:
- activation of keratinocytes
Test material
- Reference substance name:
- Carboxylic acids, di-, C5-9
- EC Number:
- 701-329-9
- Molecular formula:
- C5di: C5H8O4 C6di: C6H10O4 C7di: C7H12O4 C8di: C8H14O4 C9di: C9H16O4
- IUPAC Name:
- Carboxylic acids, di-, C5-9
Constituent 1
In vitro test system
- Details on the study design:
- TEST METHOD
This in vitro test uses human adherent HaCaT keratinocytes, an immortalized cell line. The KeratinoSens cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence.
Potential skin sensitizers are applied to the cells at 12 different concentrations for a period of 48 hours. Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase. The luciferase reporter gene is under control of a single copy of the ARE-element of the human AKR1C2. The luciferase production is measured by flash luminescence.
In parallel, cytotoxicity is measured by MTT reduction and is taken into consideration in the interpretation of the sensitisation results. This evaluation is performed in at least two independent validated runs.
TEST CELL LINE
- Type: KeratinoSens™
- Source: Givaudan, Switzerland
- Passage number: 5 in both experiments
CELL CULTURE CONDITIONS
- Type and identity of media:
Maintenance medium No. 1: Dulbecco’s Modified Eagle Medium (DMEM) containing GlutaMAX™, 1000 mg/L D-glucose and Na-Pyruvate and supplemented with 9.1% fetal calf serum (FCS) and 500 µg/mL G-418 geneticin.
Maintanence medium No. 2: DMEM with 9.1% FCS without G-418 geneticin.
Treatment medium: DMEM with 1% FCS without G-418 geneticin.
Freezing medium: DMEM with 20% FCS and 10% DMSO
TEST CONCENTRATIONS
0.20; 0.39; 0.78; 1.56; 3.13; 6.25; 12.5; 25; 50; 100; 200 and 400 μg/mL in culture medium containing 1% DMSO
CONTROLS
Vehicle control
- Substance: dimethyl sulfoxide (DMSO)
- Final concentration: 1%
Positive control
- Substance: cinnamic aldehyde
- Final concentrations: 4 - 64 µM
EXPOSURE CONDITIONS
- Exposure duration: 48 ± 2 h
- Temperature (°C): 37
- CO2 (%): 5
- relative humdity (%): 90
NUMBER OF REPLICATES: duplicates each in two independent experiments for the test item and three replicates each in two independent experiments for the negative and positive control.
DETERMINATION OF CELL VIABILITY
- Method: MTT assay
- MTT concentration: 5 mg/mL
- Incubation time: 4 h
- Device: plate reader
- Wavelength: 600 nm
DETERMINATION OF LUMINESCENCE
After incubation, the supernatants from the white assay plates were discarded. The cells were washed once with D-PBS. A volume of 20 μL of passive lysis buffer was added to each well and the cells were incubated for 20 minutes (± 2 minutes) at room temperature under orbital shaking. The plates containing the passive lysis buffer were then placed in the luminometer for reading using the following program:
- A volume of 50 μL of the luciferase substrate was added to each well,
- 1 sec after this addition, the luciferase signal was integrated for 2 seconds Luminometer with injectors and optical density reader (Varioskan Flash).
Results and discussion
- Positive control results:
- All acceptance criteria were met in both validated runs with the exception of the criterion "the average induction (Imax) in the three replicate plates for the positive control at 64 μM should be between 2 and 8" which was not fulfilled in the second validated run (i.e. Imax of 8.15). However, since a clear dose-response with increasing luciferase activity at increasing concentrations was obtained for the positive control in this run, this was considered not to have any impact on the validity of the results and the study was therefore considered as validated.
In vitro / in chemico
Resultsopen allclose all
- Key result
- Run / experiment:
- other: Experiment 1
- Parameter:
- other: maximum luciferase activity induction (Imax)
- Value:
- 1.33
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: Experiment 2
- Parameter:
- other: maximum luciferase activity induction (Imax)
- Value:
- 1.34
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- For both runs, the Imax values were < 1.5 (i.e. 1.33 and 1.34 in the first and second runs, respectively) and thus no EC1.5 was calculated. No geometric mean IC30 or IC50 was calculated since the cell viability was > 70% in both runs.
DEMONSTRATION OF TECHNICAL PROFICIENCY: not reported
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20% (17.94% in experiment 1; 15.87% in experiment 2). Therefore the negative control confirmed the validity of the study.
- Acceptance criteria met for positive control:
- Acceptance criteria met for variability between replicate measurements: The luciferase activity induced by the positive control at a concentration of 64 μM was between 2 and 8 in experiment 1 (6.71). In experiment 2, the luciferase activity was slightly above 8 (8.15). However, since a clear dose-response with increasing luciferase activity at increasing concentrations was obtained for the positive control in this run, this was considered not to have any impact on the validity of the results and the study was therefore considered as validated. The calculated EC1.5 was between 7 and 34 μM (9.38 μM in experiment 1; 6.42 μM in experiment 2).
Therefore the positive control confirmed the validity of the study.
Any other information on results incl. tables
Table 1. Evaluation of viability of culures treated with the test item
|
Concentrations (µg/mL) |
|||||||||||
Carboxylic Acids, di-, C4 -11 |
0.20 |
0.39 |
0.78 |
1.56 |
3.13 |
6.25 |
12.5 |
25 |
50 |
100 |
200 |
400 |
Viability (%) in Run 1 |
100 |
113 |
112 |
116 |
108 |
105 |
111 |
108 |
113 |
110 |
107 |
110 |
Viability (%) in Run 2 |
104 |
113 |
109 |
104 |
104 |
102 |
103 |
103 |
97 |
99 |
103 |
104 |
Mean viability (%) |
102 |
113 |
111 |
110 |
106 |
104 |
107 |
106 |
105 |
104 |
105 |
107 |
Geometric Mean (%) |
102 |
113 |
111 |
110 |
106 |
104 |
107 |
106 |
105 |
104 |
105 |
107 |
SD |
3 |
0 |
2 |
8 |
3 |
2 |
6 |
4 |
12 |
7 |
3 |
5 |
Table 2. Gene induction values, mean and SD values obtained after treatment with the test item in each run
|
Concentrations (µg/mL)
|
|||||||||||
Carboxylic Acids, di-, C4-11 |
0.20 |
0.39 |
0.78 |
1.56 |
3.13 |
6.25 |
12.5 |
25 |
50 |
100 |
200 |
400 |
Induction values in Run 1 |
1.0 |
1.1 |
1.2 |
1.1 |
1.1 |
1.1 |
1.2 |
1.2 |
1.3 |
1.1 |
1.2 |
1.3 |
Induction values in Run 2 |
1.1 |
1.0 |
1.1 |
1.1 |
1.1 |
1.0 |
1.3 |
1.2 |
1.1 |
1.3 |
1.0 |
1.1 |
Mean induction |
1.1 |
1.1 |
1.2 |
1.1 |
1.1 |
1.0 |
1.3 |
1.2 |
1.2 |
1.2 |
1.1 |
1.2 |
SD |
0.0 |
0.1 |
0.0 |
0.0 |
0.0 |
0.1 |
0.1 |
0.1 |
0.1 |
0.1 |
0.1 |
0.1 |
Applicant's summary and conclusion
- Interpretation of results:
- other: no skin sensitising potential based on the key event "activation of keratinocytes"
- Conclusions:
- There is regulatory acceptance in the EU for the application of the KeratinoSens™ test method to address key event 2, keratinocyte activation, in the skin sensitisation Adverse Outcome Pathway. Under the conditions of the test, the test item did not have a keratinocyte activating potential. The result is not conclusive with respect to the non-classification or classification as skin sensitiser of the test item and therefore further evaluation and/or data generation is required.
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