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EC number: 223-211-1 | CAS number: 3770-97-6
- 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:
- 2017-12-07 to 2018-03-20
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
- Qualifier:
- according to guideline
- Guideline:
- other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
- Type of study:
- activation of keratinocytes
Test material
- Reference substance name:
- 6-diazo-5,6-dihydro-5-oxonaphthalene-2-sulphonyl chloride
- EC Number:
- 223-211-1
- EC Name:
- 6-diazo-5,6-dihydro-5-oxonaphthalene-2-sulphonyl chloride
- Cas Number:
- 3770-97-6
- Molecular formula:
- C10H5ClN2O3S
- IUPAC Name:
- 6-(-lambda5-diazynylidene)-5-oxo-5,6-dihydronaphthalene-2-sulfonyl chloride
- Test material form:
- solid
Constituent 1
In vitro test system
- Details on the study design:
- The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.
Results and discussion
- Positive control results:
- The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (7.72 (experiment 1); 4.70 (experiment 2)).
In vitro / in chemico
Resultsopen allclose all
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: luciferase activity
- Value:
- 29.27
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 2000 µM
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: cell viability [%]
- Value:
- 3.8
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: EC1.5 [µM]
- Value:
- 526.22
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: luciferase activity
- Value:
- 16.99
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 1000 µM
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: cell viability [%]
- Value:
- 6.2
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: EC1.5 [µM]
- Value:
- 294.94
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- Acceptance Criteria
The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition.
The controls fullfilled the validity criteria of the test.
Any other information on results incl. tables
Results of the Cytotoxicity Measurement
|
Concentration [µM] |
Cell Viability [%] |
|||
Experiment 1 |
Experiment 2 |
Mean |
SD |
||
Solvent Control |
- |
100 |
100 |
100 |
0.0 |
Positive Control |
4.00 |
100.4 |
87.0 |
93.7 |
9.5 |
8.00 |
98.5 |
66.4 |
82.5 |
22.7 |
|
16.00 |
97.0 |
76.3 |
86.6 |
14.6 |
|
32.00 |
95.4 |
59.3 |
77.4 |
25.5 |
|
64.00 |
95.8 |
48.8 |
72.3 |
33.2 |
|
Test Item |
0.98 |
94.1 |
107.1 |
100.6 |
9.2 |
1.95 |
94.0 |
101.5 |
97.8 |
5.3 |
|
3.91 |
90.9 |
101.1 |
96.0 |
7.2 |
|
7.81 |
89.8 |
100.8 |
95.3 |
7.8 |
|
15.63 |
88.4 |
100.0 |
94.2 |
8.3 |
|
31.25 |
90.4 |
93.8 |
92.1 |
2.4 |
|
62.50 |
79.7 |
85.6 |
82.7 |
4.2 |
|
125.00 |
53.5 |
63.2 |
58.3 |
6.9 |
|
250.00 |
40.8 |
47.8 |
44.3 |
5.0 |
|
500.00 |
37.2 |
53.4 |
45.3 |
11.5 |
|
1000.00 |
34.8 |
6.2 |
20.5 |
20.2 |
|
2000.00 |
3.8 |
2.9 |
3.4 |
0.7 |
Induction of Luciferase Activity Experiment 1
Experiment 1 |
Concentration [µM] |
Fold Induction |
Significance |
||||
Rep. 1 |
Rep. 2 |
Rep. 3 |
Mean |
SD |
|||
Solvent Control |
- |
1.00 |
1.00 |
1.00 |
1.00 |
0.00 |
|
Positive Control |
4.00 |
1.28 |
1.23 |
1.23 |
1.25 |
0.03 |
|
8.00 |
1.44 |
1.35 |
1.29 |
1.36 |
0.07 |
|
|
16.00 |
1.82 |
1.66 |
1.50 |
1.66 |
0.16 |
* |
|
32.00 |
2.52 |
2.75 |
1.68 |
2.32 |
0.57 |
* |
|
64.00 |
7.62 |
(12.54#) |
7.82 |
7.72 |
0.10 |
* |
|
Test Item |
0.98 |
1.03 |
1.04 |
1.07 |
1.05 |
0.02 |
|
1.95 |
0.99 |
1.01 |
0.92 |
0.97 |
0.05 |
|
|
3.91 |
1.08 |
1.06 |
0.95 |
1.03 |
0.07 |
|
|
7.81 |
1.16 |
1.27 |
1.14 |
1.19 |
0.07 |
|
|
15.63 |
1.00 |
1.08 |
1.01 |
1.03 |
0.04 |
|
|
31.25 |
1.03 |
1.07 |
0.93 |
1.01 |
0.07 |
|
|
62.50 |
1.04 |
1.16 |
0.95 |
1.05 |
0.10 |
|
|
125.00 |
1.09 |
0.95 |
0.88 |
0.97 |
0.11 |
|
|
250.00 |
1.03 |
1.15 |
0.80 |
0.99 |
0.18 |
|
|
500.00 |
1.30 |
1.51 |
1.29 |
1.37 |
0.13 |
|
|
1000.00 |
3.56 |
5.19 |
3.02 |
3.92 |
1.13 |
* |
|
2000.00 |
38.70 |
33.84 |
15.27 |
29.27 |
12.37 |
* |
* = significant induction according to Student’s t-test, p < 0.05
#= outlier according to Grubbs, Nalimov and Dixon. Excluded from evaluation.
Induction of Luciferase Activity Experiment 2
Experiment 2 |
Concentration [µM] |
Fold Induction |
Significance |
||||
Rep. 1 |
Rep. 2 |
Rep. 3 |
Mean |
SD |
|||
Solvent Control |
- |
1.00 |
1.00 |
1.00 |
1.00 |
0.00 |
|
Positive Control |
4.00 |
1.16 |
1.18 |
1.10 |
1.15 |
0.04 |
|
8.00 |
1.16 |
1.10 |
1.09 |
1.12 |
0.04 |
|
|
16.00 |
1.65 |
1.69 |
1.49 |
1.61 |
0.11 |
* |
|
32.00 |
2.23 |
1.88 |
1.93 |
2.01 |
0.19 |
* |
|
64.00 |
4.84 |
4.51 |
4.75 |
4.70 |
0.17 |
* |
|
Test Item |
0.98 |
1.17 |
1.20 |
1.00 |
1.13 |
0.11 |
|
1.95 |
0.97 |
1.03 |
0.88 |
0.96 |
0.08 |
|
|
3.91 |
0.97 |
0.91 |
0.99 |
0.96 |
0.04 |
|
|
7.81 |
0.87 |
0.92 |
0.82 |
0.87 |
0.05 |
|
|
15.63 |
0.88 |
0.98 |
1.07 |
0.98 |
0.09 |
|
|
31.25 |
0.87 |
0.81 |
0.81 |
0.83 |
0.04 |
|
|
62.50 |
0.93 |
0.95 |
0.78 |
0.89 |
0.09 |
|
|
125.00 |
1.09 |
1.06 |
0.85 |
1.00 |
0.13 |
|
|
250.00 |
1.27 |
1.04 |
0.96 |
1.09 |
0.16 |
|
|
500.00 |
6.05 |
2.42 |
1.64 |
3.37 |
2.35 |
|
|
1000.00 |
9.30 |
29.65 |
12.01 |
16.99 |
11.05 |
* |
|
2000.00 |
0.01 |
0.01 |
1.52 |
0.51 |
0.87 |
|
* = significant induction according to Student’s t-test, p < 0.05
Induction of Luciferase Activity – Overall Induction
|
Concentration [µM] |
Fold Induction |
Significance |
|||
Experiment 1 |
Experiment 2 |
Mean |
SD |
|||
Solvent Control |
- |
1.00 |
1.00 |
1.00 |
0.00 |
|
Positive Control |
4.00 |
1.25 |
1.15 |
1.20 |
0.07 |
|
8.00 |
1.36 |
1.12 |
1.24 |
0.17 |
|
|
16.00 |
1.66 |
1.61 |
1.63 |
0.04 |
* |
|
32.00 |
2.32 |
2.01 |
2.16 |
0.22 |
* |
|
64.00 |
9.33 |
4.70 |
7.01 |
3.27 |
|
|
Test Item |
0.98 |
1.05 |
1.13 |
1.09 |
0.06 |
|
1.95 |
0.97 |
0.96 |
0.97 |
0.01 |
|
|
3.91 |
1.03 |
0.96 |
0.99 |
0.05 |
|
|
7.81 |
1.19 |
0.87 |
1.03 |
0.22 |
|
|
15.63 |
1.03 |
0.98 |
1.00 |
0.04 |
|
|
31.25 |
1.01 |
0.83 |
0.92 |
0.13 |
|
|
62.50 |
1.05 |
0.89 |
0.97 |
0.12 |
|
|
125.00 |
0.97 |
1.00 |
0.99 |
0.02 |
|
|
250.00 |
0.99 |
1.09 |
1.04 |
0.07 |
|
|
500.00 |
1.37 |
3.37 |
2.37 |
1.42 |
|
|
1000.00 |
3.92 |
16.99 |
10.45 |
9.24 |
|
|
2000.00 |
29.27 |
0.51 |
14.89 |
20.33 |
|
* = significant induction according to Student’s t-test, p < 0.05
Additional Parameters
Parameter |
Experiment 1 |
Experiment 2 |
Mean |
SD |
EC1.5[µM] |
526.22 |
294.94 |
410.58 |
163.54 |
Imax |
29.27 |
16.99 |
23.13 |
8.69 |
IC30[µM] |
85.61 |
106.04 |
95.82 |
14.45 |
IC50[µM] |
159.32 |
231.00 |
195.16 |
50.69 |
Acceptance Criteria
Criterion |
Range |
Experiment 1 |
pass/fail |
Experiment 2 |
pass/fail |
CV Solvent Control |
< 20% |
13.1 |
pass |
9.6 |
pass |
No. of positive control concentration steps with significant luciferase activity induction >1.5 |
≥ 1 |
3.0 |
pass |
3.0 |
pass |
EC1.5 PC |
7 < x < 34 µM |
11.72 |
pass |
14.23 |
pass |
Induction PC at 64 µM |
2.00 < x < 8.00 |
7.72* |
pass |
4.70 |
pass |
* = Mean out of replicate 1 and 3. 2 replicate is an outlier according to Grubbs, Nalimov and Dixon.
Historical Data
Acceptance Criterion |
Range |
Mean |
SD |
N |
CV Solvent Control |
< 20% |
11.3 |
3.3 |
41 |
No. of positive control concentration steps with significant luciferase activity induction >1.5 |
≥ 1 |
2.3 |
0.6 |
41 |
EC1.5 PC |
7 < x < 34 µM |
20.4 |
6.7 |
41 |
Induction PC at 64 µM |
2.00 < x < 8.00 |
3.3 |
1.1 |
41 |
Applicant's summary and conclusion
- Interpretation of results:
- other: should be considered in the context of integrated approached such as IATA
- Conclusions:
- In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA. - Executive summary:
In the present study the test material was dissolved in DMSO.
Based on a molecular weight of 268.5 g/mol a stock solution of 200 mM was prepared.
Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:
2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM
Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.
In the first experiment, a max luciferase activity (Imax) induction of 29.27 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 3.8%. The lowest tested concentration with a significant luciferase induction >1.5 (3.92) was found to be 1000 µM. The corresponding cell viability was <70% (34.8%).The calculated EC1.5 was <1000 µM (526.22)
In the second experiment, a max luciferase activity (Imax) induction of 16.99 was determined at a test item concentration of 1000 µM. The corresponding cell viability was 6.2%. The lowest tested concentration with a significant luciferase induction >1.5 (3.37) was found to be 500 µM. The corresponding cell viability was <70% (53.4%).The calculated EC1.5 was < 1000 µM (294.94).
No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.
Under the condition of this study the test item is therefore considered as non sensitiser.
The luciferase activity induced by the positive control at a concentration of 64 µM should be between 2 and 8. In the first experiment one of the technical replicate exceeded the threshold with a fold induction of 12.54. On the one hand, the positive control clearly induces the luciferase induction in the KerationSens™ cell line, showing the capability of the test system to predict sensitisation. On the other hand, the results in this first experiment are clearly negative, indicating no oversensitivity of the test system. The single value of the positive control exceeding the upper threshold was therefore considered to be an outliner and not biological relevant. Furthermore all other controls confirmed the validity of the study
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