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EC number: 204-310-9 | CAS number: 119-27-7
- 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:
- key study
- Study period:
- 27 November - 21 December, 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guideline
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- activation of keratinocytes
- Justification for non-LLNA method:
- Skin sensitisers have been reported to induce genes that are regulated by the antioxidant response element (ARE). The KeratinoSensTM test is a method for which validation studies have been completed followed by an independent peer review conducted by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM). The KeratinoSensTM test method was considered scientifically valid to be used as part of an IATA (Integrated Approach to Testing and Assessment), to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling. The method cannot be used on its own, neither to sub-categorise skin sensitisers into subcategories IA and 1B as defined by the UN GHS, for authorities implementing these two optional subcategories, nor to predict potency for safety assessment decisions. However, depending on the regulatory framework a positive result may be used on its own to classify a chemical into UN GHS category 1.
Test material
- Reference substance name:
- 2,4-dinitroanisole
- EC Number:
- 204-310-9
- EC Name:
- 2,4-dinitroanisole
- Cas Number:
- 119-27-7
- Molecular formula:
- C7H6N2O5
- IUPAC Name:
- 2,4-dinitroanisole
Constituent 1
- Specific details on test material used for the study:
- Supplier Sponsor
Test Item name DNAN (2,4-Dinitroanisole)
Supplier Code CAS No. 119-27-7
Supplier batch/lot number 55503625 Lot 02/13
CAS number 119-27-7
Molecular Weight 198
Purity
Expiry Date 28 April 2019
Physical state Solid
Storage Conditions Room Temperature, Dark
In vitro test system
- Details on the study design:
- Description of the test system:
The KeratinoSensTM cell line (test system) is an immortalized adherent cell line derived from HaCaT human keratinocytes, stably transfected with a selectable plasmid containing the luciferase gene under the transcriptional control of the Anti-oxidant Response Element (ARE) from a gene that is known to be upregulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances.
Method of administration of test item:
Per plate, single application of 12 concentrations of the test item was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1%. The top concentration was previously determined by solubility testing.
Method of administration of reference items:
Per plate, single application of 5 concentrations of the positive control was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1% and single application of culture medium with 1% DMSO was applied as the negative control (6 wells per plate). One well per plate is left empty (no cells).
Exposure times of test items and reference items:
Cells were incubated with the test or reference item for 48 ± 2h before endpoints measurements.
Number of repetitions:
Three repetitions (runs) were performed. Each repetition consisted of 3 x 96-well plates for luminescence and 2 x 96-well plates for MTT.
Overview
Preliminary testing: Determination of the top concentration by solubility testing
Day 1: Seeding cells (3 x 96-well plates for Luminescence; 2 x 96-well plate for MTT); 10,000 cells per well, 14.
Day 2: 24h after seeding the test and control items were applied and plates were incubated at 370C, 5% C02, 2 95% relative humidity for 48 ± 2h.
Day 4: Evaluation of luciferase activity by luminescence (3 plates) and cell viability by MIT testing (2 plates)
Data Analysis
XCellR8 Forms F0056 A (version 01) and B (version 04): Data Analysis for KeratinoSensTM were used to analyse data. These forms are Microsoft Excel workbooks containing formulae to process the raw data as described in SOP L0057. The spreadsheets have been validated in-house (July 2017).
The following parameters were calculated in the KeratinoSensTM test method:
the maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the test item and positive control;
the EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5- fold threshold (i.e. 50% enhanced luciferase activity);
For each concentration showing > 1.5-fold luciferase activity induction, statistical significance is calculated (e.g. by a two-tailed Student's t-test), comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). The lowest concentration with > 1.5-fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.
The percentage of viability as compared to the Negative control
Results and discussion
- Positive control results:
- Acceptance Criteria (Mean of three Replicates)
Criteria Result Pass or Fail
I-Positive Control (PC) (Cinnamic aldehyde) induction 21.5-fold in at least one
concentration Yes Pass
2-Average induction of PC at 32gM is [1.6-3.0] Yes (2.11) Pass
3-EC1.5value is [6-39wM] Yes (11.50) Pass
4-CV% of blank values < 20% Yes (13.06%) Pass
In vitro / in chemico
Results
- Key result
- Run / experiment:
- other: Mean value
- Parameter:
- other: EC1.5
- Value:
- 1.5
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
Any other information on results incl. tables
Table 1 Sensitisation potential of the test item - Repetition 1
Rep 1 |
Test Item Concentration (µM) |
|||||||||||
0.977 |
1.953 |
3.906 |
7.813 |
15.625 |
31.250 |
62.5 |
125 |
250 |
500 |
1000 |
2000 |
|
Mean of fold induction |
0.946 |
0.969 |
1.111 |
1.105 |
1.032 |
1.089 |
1.119 |
1.173 |
1.134 |
1.364 |
1.480 |
1.412 |
SD |
0.090 |
0.146 |
0.128 |
0.055 |
0.042 |
0.062 |
0.082 |
0.124 |
0.079 |
0.081 |
0.257 |
0.235 |
Viability % |
112.22 |
97.99 |
103.41 |
95.06 |
90.00 |
86.92 |
102.53 |
96.54 |
88.83 |
100.01 |
112.47 |
101.28 |
Imax |
1.480 at 1000µM |
|||||||||||
EC1.5 |
No EC1.5 as no concentration did induce the luciferase activity above the 1.5 threshold |
|||||||||||
IC50 IC30 |
>2000µM as viability at 2000µM was 101.28% |
|||||||||||
>2000µM as viability at 2000µM was 101.28% |
Table 2 Sensitisation potential of the test item - Repetition 2
Rep 2 |
Test item concentration (µM) |
||||||||||||
0.977 |
1.953 |
3.906 |
7.813 |
15.625 |
31.250 |
62.5 |
125 |
250 |
500 |
1000 |
2000 |
||
Mean of fold induction |
1.027 |
1.150 |
1.118 |
1.110 |
0.969 |
1.191 |
1.245 |
1.237 |
1.358 |
1.861 |
2.422 |
1.067 |
|
SD |
0.045 |
0.135 |
0.084 |
0.084 |
0.051 |
0.087 |
0.090 |
0.067 |
o. 165 |
0.078 |
0.247 |
0.134 |
|
Viability % |
107.44 |
99.62 |
105.76 |
101.36 |
103.09 |
110.01 |
111.12 |
99.59 |
108.54 |
118.30 |
134.28 |
138.56 |
|
u' Imax |
2.422 at 1000µM |
||||||||||||
EC1.5 |
320.67 |
||||||||||||
IC50 |
|
>2000µM as viability at 2000µM was 138.56% |
|||||||||||
IC30 |
>2000µM as viability at 2000µM was 138.56% |
Table 3 Sensitisation potential of the test item - Repetition 3
Rep 3 |
Test item concentration (µM) |
||||||||||||
0.977 |
1.953 |
3.906 |
7.813 |
15.625 |
31.250 |
62.5 |
125 |
250 |
500 |
1000 |
2000 |
||
Mean of fold induction |
0.795 |
0.839 |
0.933 |
0.983 |
0.895 |
0.949 |
0.977 |
1.130 |
0.954 |
1.447 |
0.958 |
||
SD |
0.032 |
0.073 |
0.004 |
0.105 |
0.127 |
0.062 |
0.049 |
0.085 |
0.145 |
0.008 |
0.153 |
0.007 |
|
Viability % |
96.81 |
99.79 |
102.82 |
107.08 |
98.47 |
101.54 |
106.68 |
96.28 |
101.57 |
112.28 |
129.11 |
165.15 |
|
Imax |
1.447 at IOOOµM |
||||||||||||
EC1.5 |
No EC1.5 as no concentration did induce the luciferase activity above the 1.5 threshold |
||||||||||||
IC50 |
|
>2000µM as viability at 2000µM was 165.15% |
|||||||||||
IC30 |
|
>2000µM as viability at 2000µM was 165.15% |
Table 4 Determination criteria for the skin sensitisation potential of DNAN
REP1 |
REP2 |
REP3 |
|
Does at least one concentation of Test Item induce luciferase activity 21.5-fold: |
No |
Yes |
No |
Does the first concentration inducing luciferase activity above 1.5, have a viability above 70%: |
N/A |
Yes |
N/A |
Does EC1.5 value occur at a concentration <IOOOµM (or <200µg/mI) |
N/A |
Yes |
N/A |
Does the test item induce the luciferase in a dosedependent manner |
N/A |
Yes |
N/A |
Classification |
Non-Sensitiser |
Sensitiser |
Non-Sensitiser |
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- In this study, DNAN was classified as a non-sensitiser to human skin.
- Executive summary:
The human skin sensitisation potential of DNAN was assessed using the validated in vitro method, the KeratinoSensTMassay, adapted to fully animal-free by XCellR8, and validated in-house to determine keratinocyte activation.
After 48h exposure of cells with 12 concentrations of DNAN, Luciferase measurements and MIT viability testing were performed.
In this study, DNAN was classified as a non-sensitiser.
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