Registration Dossier
Registration Dossier
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EC number: 295-173-4 | CAS number: 91845-03-3
Skin sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2019-07-18 to 2019-09-06
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP 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
Test material
- Reference substance name:
- Fatty acids, C12-18 and C18-unsatd., reaction products with N,N-dimethyl-1,3-propanediamine and triethanolamine
- Cas Number:
- 91845-03-3
- Molecular formula:
- Unknown
- IUPAC Name:
- Fatty acids, C12-18 and C18-unsatd., reaction products with N,N-dimethyl-1,3-propanediamine and triethanolamine
- Test material form:
- solid: bulk
Constituent 1
- Specific details on test material used for the study:
- Name: 35760/270
Chemical Name: Fatty acids, C12-18 and C18-unsatd., reaction products with N,N-dimethyl-1,3-propanediamine and triethanolamin
Batch No.: 19953859
CAS No.: 91845-03-3
Molecular Weight: not applicable
Purity: 100% (UVCB)
Physical State: solid
Colour: beige
Log KOW not specified by the sponsor
Stability: stable
Storage Conditions: at room temperature, protected from light
Expiry Date: 16 January 2020
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.
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 (5.0 (experiment 1); 2.52 (experiment 2) and 3.54 (experiment 3)).
In vitro / in chemico
Resultsopen allclose all
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: luciferase activity
- Value:
- 3.78
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 7.81 µM
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: cell viability [%]
- Value:
- 120
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: EC1.5 [µM]
- Value:
- 4.58
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other:
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: luciferase activity
- Value:
- 1.57
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 7.81 µM
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: cell viability [%]
- Value:
- 64.4
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: EC1.5 [µM]
- Value:
- 7.48
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other:
- Run / experiment:
- other: 3
- Parameter:
- other: luciferase activity
- Value:
- 2.75
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 7.81 µM
- Run / experiment:
- other: 3
- Parameter:
- other: cell viability [%]
- Value:
- 96.9
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Run / experiment:
- other: 3
- Parameter:
- other: EC1.5 [µM]
- Value:
- 5.19
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other:
- 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 |
Experiment 3 |
Mean |
SD |
||
Solvent Control (DMSO) |
- |
100 |
100 |
100 |
100 |
0.0 |
Positive Control |
4.00 |
100.1 |
97.4 |
90.0 |
95.8 |
5.2 |
8.00 |
105.5 |
102.7 |
97.5 |
101.9 |
4.0 |
|
16.00 |
99.4 |
103.4 |
101.0 |
101.3 |
2.0 |
|
32.00 |
100.0 |
98.2 |
100.1 |
99.4 |
1.1 |
|
64.00 |
102.2 |
102.3 |
102.3 |
102.2 |
0.0 |
|
Solvent Control (THF) |
- |
100 |
100 |
100 |
100 |
0.0 |
Test Item |
0.98 |
99.0 |
85.8 |
103.8 |
96.2 |
9.3 |
1.95 |
74.8 |
97.2 |
83.8 |
85.2 |
11.3 |
|
3.91 |
76.2 |
74.1 |
87.9 |
79.4 |
7.4 |
|
7.81 |
120.0 |
64.4 |
96.9 |
93.8 |
28.0 |
|
15.63 |
26.3 |
20.3 |
21.4 |
22.7 |
3.2 |
|
31.25 |
1.3 |
0.7 |
-1.2 |
0.3 |
1.3 |
|
62.50 |
1.5 |
0.6 |
-1.1 |
0.3 |
1.3 |
|
125.00 |
1.4 |
0.6 |
-0.8 |
0.4 |
1.1 |
|
250.00 |
1.4 |
0.5 |
-0.6 |
0.5 |
1.0 |
|
500.00 |
1.1 |
0.5 |
-0.9 |
0.2 |
1.1 |
|
1000.00 |
1.1 |
0.8 |
-1.0 |
0.3 |
1.1 |
|
2000.00 |
1.1 |
1.0 |
-0.1 |
0.7 |
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 (DMSO) |
- |
1.00 |
1.00 |
1.00 |
1.00 |
0.00 |
|
Positive Control |
4.00 |
1.30 |
1.16 |
1.15 |
1.21 |
0.08 |
|
8.00 |
1.02 |
1.18 |
1.07 |
1.09 |
0.08 |
|
|
16.00 |
1.50 |
1.54 |
1.45 |
1.50 |
0.04 |
|
|
32.00 |
2.11 |
2.75 |
2.01 |
2.29 |
0.40 |
* |
|
64.00 |
5.29 |
5.22 |
4.49 |
5.00 |
0.44 |
* |
|
Solvent Control (THF) |
- |
1.00 |
1.00 |
1.00 |
1.00 |
0.00 |
|
Test Item |
0.98 |
1.11 |
1.16 |
1.16 |
1.14 |
0.03 |
|
1.95 |
0.91 |
0.99 |
1.08 |
0.99 |
0.09 |
|
|
3.91 |
1.09 |
0.97 |
1.01 |
1.02 |
0.07 |
|
|
7.81 |
3.90 |
4.07 |
3.38 |
3.78 |
0.36 |
* |
|
15.63 |
2.25 |
2.88 |
2.70 |
2.61 |
0.33 |
* |
|
31.25 |
0.01 |
0.00 |
0.01 |
0.01 |
0.00 |
|
|
62.50 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
|
125.00 |
0.00 |
0.00 |
0.00 |
0.00 |
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 |
0.01 |
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 (DMSO) |
- |
1.00 |
1.00 |
1.00 |
1.00 |
0.00 |
|
Positive Control |
4.00 |
0.85 |
0.99 |
1.07 |
0.97 |
0.11 |
|
8.00 |
0.95 |
1.21 |
1.08 |
1.08 |
0.13 |
|
|
16.00 |
1.18 |
1.32 |
1.17 |
1.22 |
0.08 |
|
|
32.00 |
1.57 |
1.64 |
1.48 |
1.56 |
0.08 |
* |
|
64.00 |
2.26 |
2.84 |
2.45 |
2.52 |
0.29 |
* |
|
Solvent Control (THF) |
- |
1.00 |
1.00 |
1.00 |
1.00 |
0.00 |
|
Test Item |
0.98 |
0.84 |
1.25 |
1.12 |
1.07 |
0.21 |
|
1.95 |
0.83 |
0.94 |
0.89 |
0.89 |
0.06 |
|
|
3.91 |
0.65 |
0.80 |
0.77 |
0.74 |
0.08 |
|
|
7.81 |
1.39 |
1.76 |
1.56 |
1.57 |
0.19 |
* |
|
15.63 |
1.13 |
1.24 |
1.06 |
1.14 |
0.09 |
|
|
31.25 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
|
62.50 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
|
125.00 |
0.00 |
0.00 |
0.00 |
0.00 |
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 Experiment 3
Experiment 3 |
Concentration [µM] |
Fold Induction |
Significance |
||||
Rep. 1 |
Rep. 2 |
Rep. 3 |
Mean |
SD |
|||
Solvent Control (DMSO) |
- |
1.00 |
1.00 |
1.00 |
1.00 |
0.00 |
|
Positive Control |
4.00 |
0.97 |
1.12 |
1.07 |
1.05 |
0.08 |
|
8.00 |
1.06 |
1.20 |
1.29 |
1.19 |
0.12 |
||
16.00 |
1.28 |
1.39 |
1.39 |
1.35 |
0.07 |
||
32.00 |
1.53 |
1.59 |
1.75 |
1.63 |
0.11 |
* |
|
64.00 |
3.19 |
3.27 |
4.15 |
3.54 |
0.54 |
* |
|
Solvent Control (THF) |
- |
1.00 |
1.00 |
1.00 |
1.00 |
0.00 |
|
Test Item |
0.98 |
1.14 |
1.44 |
1.03 |
1.21 |
0.21 |
|
1.95 |
1.00 |
1.32 |
0.95 |
1.09 |
0.20 |
|
|
3.91 |
0.79 |
0.95 |
0.93 |
0.89 |
0.09 |
|
|
7.81 |
2.50 |
2.99 |
2.76 |
2.75 |
0.24 |
* |
|
15.63 |
2.32 |
2.63 |
3.48 |
2.81 |
0.60 |
* |
|
31.25 |
0.16 |
0.30 |
0.06 |
0.17 |
0.12 |
|
|
62.50 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
|
125.00 |
0.00 |
0.00 |
0.00 |
0.00 |
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
Overall Induction |
Concentration [µM] |
Fold Induction |
Significance |
||||
Experiment 1 |
Experiment 2 |
Experiment 3 |
Mean |
SD |
|||
Solvent Control (DMSO) |
- |
1.00 |
1.00 |
1.00 |
1.00 |
0.00 |
|
Positive Control |
4.00 |
1.21 |
0.97 |
1.05 |
1.08 |
0.12 |
|
8.00 |
1.09 |
1.08 |
1.19 |
1.12 |
0.06 |
|
|
16.00 |
1.50 |
1.22 |
1.35 |
1.36 |
0.14 |
|
|
32.00 |
2.29 |
1.56 |
1.63 |
1.83 |
0.40 |
* |
|
64.00 |
5.00 |
2.52 |
3.54 |
3.68 |
1.25 |
* |
|
Solvent Control (THF) |
- |
1.00 |
1.00 |
1.00 |
1.00 |
0.00 |
|
Test Item |
0.98 |
1.14 |
1.07 |
1.21 |
1.14 |
0.07 |
|
1.95 |
0.99 |
0.89 |
1.09 |
0.99 |
0.10 |
|
|
3.91 |
1.02 |
0.74 |
0.89 |
0.89 |
0.14 |
|
|
7.81 |
3.78 |
1.57 |
2.75 |
2.70 |
1.11 |
|
|
15.63 |
2.61 |
1.14 |
2.81 |
2.19 |
0.91 |
|
|
31.25 |
0.01 |
0.00 |
0.17 |
0.06 |
0.10 |
|
|
62.50 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
|
125.00 |
0.00 |
0.00 |
0.00 |
0.00 |
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
Additional Parameters
Parameter |
Experiment 1 |
Experiment 2 |
Experiment 3 |
Mean |
SD |
EC1.5 |
4.58 |
7.48 |
5.19 |
5.75 |
1.53 |
Imax |
3.78 |
1.57 |
2.81 |
2.72 |
1.11 |
IC30 |
11.98 |
5.56 |
10.60 |
9.38 |
3.38 |
IC50 |
13.65 |
10.37 |
12.67 |
12.23 |
1.69 |
Acceptance Criteria
Criterion |
Range |
Experiment 1 |
pass / fail |
Experiment 2 |
pass / fail |
Experiment 3 |
pass / fail |
CV Solvent Control PC (1% DMSO) |
< 20% |
8.1 |
pass |
15.7 |
pass |
9.9 |
pass |
CV Solvent Control TI (1% THF) |
< 20% |
10.8 |
pass |
15.5 |
pass |
17.8 |
pass |
No. of positive control concentration steps with significant luciferase activity induction >1.5 |
≥ 1 |
2.0 |
pass |
2.0 |
pass |
2.0 |
pass |
EC1.5 PC [µM] |
7 < x < 34 µM |
16.07 |
pass |
29.03 |
pass |
24.64 |
pass |
Induction PC at 64 µM |
2 .00 < x < 8.00 |
5.00 |
pass |
2.52 |
pass |
3.54 |
pass |
Historical Data
Acceptance Criterion |
Range |
Mean |
SD |
N |
CV Solvent Control |
< 20% |
11.6 |
3.5 |
96 |
No. of positive control concentration steps with significant luciferase activity induction >1.5 |
≥ 1 |
2.4 |
0.6 |
96 |
EC1.5 PC |
7 < x < 34 µM |
18.5 |
6.0 |
96 |
Induction PC at 64 µM |
2.00 < x < 8.00 |
3.8 |
1.5 |
96 |
Applicant's summary and conclusion
- Conclusions:
- In this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs, but without any dose response. Therefore, the test item has to be considered as inconclusive.
The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP. - Executive summary:
In the present study 35760/270 was dissolved in THF. As the test item had no defined molecular weight, the test was performed using a pro forma molecular weight of 200 g/mol. Based on this, 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 3.78 was determined at a test item concentration of 7.81 µM. The corresponding cell viability was 120%. The calculated EC1.5 was <1000 µM (4.58 µM).
In the second experiment, a max luciferase activity (Imax) induction of 1.57 was determined at a test item concentration of 7.81 µM. The corresponding cell viability was 64.4%. This was the only tested concentration with a significant luciferase induction >1.5. The calculated EC1.5 was <1000 µM (7.48 µM).
In the third experiment, a max luciferase activity (Imax) induction of 2.81 was determined at a test item concentration of 15.63 µM. The corresponding cell viability was 21.4%. The calculated EC1.5 was <1000 µM (5.19 µM).
Due to a missing dose response in the first and third main experiment the test item has to be considered as inconclusive.
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.
