Registration Dossier
Registration Dossier
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 202-928-3 | CAS number: 101-25-7
Skin sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2019-05-08 to 2019-05-20
- 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 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
- Justification for non-LLNA method:
- The KeratinoSens™ assay is supposed to address the second key event of the skin sensitisation process as defined by the adverse outcome pathway (AOP), the induction of cyto-protective signalling pathways in keratinocytes in response to electrophiles and oxidative stress. The KeratinoSens™ assay addresses the effect on the antioxidant response element (ARE)-dependent pathway in the KeratinoSens™ cell line by measuring the induction of an ARE dependent gene product, the luciferase gene. The luciferase gene induction following exposure to test chemicals is measured in cell lysates by luminescence detection, allowing the discrimination between sensitisers and non-sensitisers.
Since activation of the Keap1-Nrf2-ARE pathway addresses only the second key event of the skin sensitisation AOP, information from test methods based on the activation of this pathway is unlikely to be sufficient when used on its own to conclude on the skin sensitisation potential of chemicals. Therefore data generated according to OECD 442D should be considered in the context of integrated approaches, such as IATA, combining them with other complementary information e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.
Test material
- Reference substance name:
- 3,7-dinitroso-1,3,5,7-tetraazabicyclo[3.3.1]nonane
- EC Number:
- 202-928-3
- EC Name:
- 3,7-dinitroso-1,3,5,7-tetraazabicyclo[3.3.1]nonane
- Cas Number:
- 101-25-7
- Molecular formula:
- C5H10N6O2
- IUPAC Name:
- 3,7-dinitroso-1,3,5,7-tetraazabicyclo[3.3.1]nonane
- Test material form:
- solid
- Details on test material:
- - Name: 3,7-Dinitroso-1,3,5,7-tetraazabicyclo[3.3.1]nonane
- CAS: 101-25-7
- Batch: 458231 GDSK
- MW: 186.17 g/mol
- Purity: 95.5 %
- Appearance: beige solid
- Stability: instable after repeated contact to light; undergoes hydrolysis at acidic pH
- Expiry Date: 31 July 2019
- Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.
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 (2,78 (experiment 1); 3,28 (experiment 2)).
In vitro / in chemico
Resultsopen allclose all
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: luciferase activity
- Remarks:
- I max
- Value:
- 1.15
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration 62.50 µM
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: cell viability [%]
- Value:
- 97.8
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration 62.50 µM
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: EC1.5 [µM]
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- not determinable
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: luciferase activity
- Remarks:
- I max
- Value:
- 1.13
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration 62.50 µM
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: cell viability [%]
- Value:
- 98.6
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration 62.50 µM
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: EC1.5 [µM]
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- not determinable
- Other effects / acceptance of results:
- All acceptance criteria were fullfilled proving the validity of the test.
Any other information on results incl. tables
In the first experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. From the concentration 125 μM onwards the test item was cytotoxic.
In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. From the concentration 125 μM onwards the test item was cytotoxic.
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.
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 |
99.5 |
97.1 |
|
98.3 |
1.7 |
8.00 |
103.7 |
101.6 |
|
102.6 |
1.5 |
|
16.00 |
106.3 |
101.9 |
|
104.1 |
3.1 |
|
32.00 |
104.8 |
105.6 |
|
105.2 |
0.5 |
|
64.00 |
107.8 |
103.5 |
|
105.6 |
3.1 |
|
Test Item |
0.98 |
97.4 |
109.9 |
|
103.7 |
8.9 |
1.95 |
96.4 |
106.2 |
|
101.3 |
6.9 |
|
3.91 |
97.1 |
102.1 |
|
99.6 |
3.5 |
|
7.81 |
95.0 |
99.8 |
|
97.4 |
3.4 |
|
15.63 |
96.0 |
104.5 |
|
100.3 |
6.0 |
|
31.25 |
92.1 |
98.2 |
|
95.1 |
4.3 |
|
62.50 |
97.8 |
98.6 |
|
98.2 |
0.6 |
|
125.00 |
0.3 |
0.1 |
|
0.2 |
0.2 |
|
250.00 |
2.6 |
2.2 |
|
2.4 |
0.3 |
|
500.00 |
1.7 |
0.4 |
|
1.1 |
0.9 |
|
1000.00 |
0.3 |
0.3 |
|
0.3 |
0.0 |
|
2000.00 |
-0.1 |
0.3 |
|
0.1 |
0.3 |
|
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 |
1.17 |
1.11 |
1.12 |
1.13 |
0.04 |
|
8.00 |
1.33 |
1.62 |
1.22 |
1.39 |
0.21 |
|
|
16.00 |
1.44 |
1.36 |
1.22 |
1.34 |
0.11 |
|
|
32.00 |
1.70 |
1.93 |
1.54 |
1.72 |
0.20 |
* |
|
64.00 |
2.79 |
3.07 |
2.47 |
2.78 |
0.30 |
* |
|
Test Item |
0.98 |
1.03 |
1.10 |
1.14 |
1.09 |
0.06 |
|
1.95 |
0.96 |
1.07 |
1.03 |
1.02 |
0.05 |
|
|
3.91 |
1.00 |
1.05 |
0.94 |
1.00 |
0.06 |
|
|
7.81 |
0.98 |
1.05 |
0.98 |
1.00 |
0.04 |
|
|
15.63 |
1.01 |
1.00 |
0.94 |
0.98 |
0.04 |
|
|
31.25 |
1.11 |
1.20 |
0.99 |
1.10 |
0.10 |
|
|
62.50 |
1.08 |
1.19 |
1.18 |
1.15 |
0.06 |
|
|
125.00 |
0.11 |
0.12 |
0.11 |
0.11 |
0.01 |
|
|
250.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
|
500.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
|
1000.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
|
2000.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
|
* = significant induction according to Student’s t-test, p<0.05
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 |
1.35 |
1.22 |
1.94 |
1.50 |
0.39 |
|
8.00 |
1.33 |
1.49 |
1.29 |
1.37 |
0.10 |
|
|
16.00 |
1.66 |
1.87 |
1.60 |
1.71 |
0.14 |
* |
|
32.00 |
2.29 |
1.85 |
2.24 |
2.13 |
0.24 |
* |
|
64.00 |
3.57 |
3.46 |
2.80 |
3.28 |
0.42 |
* |
|
Test Item |
0.98 |
1.16 |
1.32 |
0.88 |
1.12 |
0.22 |
|
1.95 |
1.11 |
0.91 |
0.88 |
0.97 |
0.12 |
|
|
3.91 |
0.88 |
0.92 |
0.87 |
0.89 |
0.03 |
|
|
7.81 |
0.91 |
0.99 |
0.82 |
0.91 |
0.09 |
|
|
15.63 |
0.90 |
0.90 |
0.84 |
0.88 |
0.04 |
|
|
31.25 |
0.93 |
0.88 |
0.88 |
0.90 |
0.03 |
|
|
62.50 |
1.09 |
1.25 |
1.04 |
1.13 |
0.11 |
|
|
125.00 |
0.01 |
0.00 |
0.01 |
0.01 |
0.00 |
|
|
250.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
|
500.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
|
1000.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
|
2000.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
|
* = significant induction according to Student’s t-test, p<0.05
Induction of Luciferase Activity – Overall Induction
|
Concentration [µM] |
Fold Induction |
|||
Experiment 1 |
Experiment 2 |
Mean |
SD |
||
Solvent Control |
- |
1.00 |
1.00 |
1.00 |
0.00 |
Positive Control |
4.00 |
1.13 |
1.50 |
1.32 |
0.26 |
8.00 |
1.39 |
1.37 |
1.38 |
0.01 |
|
16.00 |
1.34 |
1.71 |
1.53 |
0.26 |
|
32.00 |
1.72 |
2.13 |
1.93 |
0.28 |
|
64.00 |
2.78 |
3.28 |
3.03 |
0.35 |
|
Test Item |
0.98 |
1.09 |
1.12 |
1.11 |
0.02 |
1.95 |
1.02 |
0.97 |
0.99 |
0.04 |
|
3.91 |
1.00 |
0.89 |
0.95 |
0.07 |
|
7.81 |
1.00 |
0.91 |
0.96 |
0.07 |
|
15.63 |
0.98 |
0.88 |
0.93 |
0.07 |
|
31.25 |
1.10 |
0.90 |
1.00 |
0.15 |
|
62.50 |
1.15 |
1.13 |
1.14 |
0.02 |
|
125.00 |
0.11 |
0.01 |
0.06 |
0.07 |
|
250.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
500.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
1000.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
2000.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
* = significant induction according to Student’s t-test, p<0.05
Additional Parameters
Parameter |
Experiment 1 |
Experiment 2 |
Mean |
SD |
EC1.5 |
n.a. |
n.a. |
n.a. |
n.a. |
Imax |
1.15 |
1.13 |
1.14 |
0.02 |
IC30 |
80.31 |
80.64 |
80.48 |
0.24 |
IC50 |
93.14 |
93.33 |
93.23 |
0.14 |
IC70 |
105.96 |
106.02 |
105.99 |
0.04 |
n.a. = not applicable
Applicant's summary and conclusion
- Interpretation of results:
- other: Negative in the KeratinoSens Assay
- 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 item was dissolved in DMSO. Based on a molecular weight of 186.17 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, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. From the concentration 125 µM onwards the test item was cytotoxic.
In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. From the concentration 125 µM onwards the test item was cytotoxic.
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.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
