N-[3-(dimethylamino)propyl]dodecanamide N-oxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 - 19 Jun 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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 in 2018

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

N-[3-(dimethylamino)propyl]dodecanamide N-oxide

EC Number:

263-218-7

EC Name:

N-[3-(dimethylamino)propyl]dodecanamide N-oxide

Cas Number:

61792-31-2

Molecular formula:

C17H36N2O2

IUPAC Name:

N-[3-(dimethylamino)propyl]dodecanamide N-oxide

In vitro test system

Details on the study design:

TEST CELL LINE: KeratinoSensTM
- Cell type: HaCaT cells (human keratinocytes)
- Source: Givaudan (Duebendorf, Switzerland)
- Passage number: 11 (Experiment I) and 14 (Experiment II)

TEST METHOD
The in vitro KeratinoSensTM assay enables the detection of the skin sensitizing potential of a test item by analysing the activation of keratinocytes. This activation step represents the second molecular key event of the adverse outcome pathway, which is the induction of cyto-protective signaling pathways in keratinocytes in response to electrophiles and oxidative stress. The KeratinoSens assay addresses the effect on the antioxidant response element (ARE) Nrf2-dependent pathway in the transgenic KeratinoSens cell line, which stably expresses the ARE-Nrf2-dependet luciferase gene. The Nrf2-dependent induction of this reporter gene is analysed upon exposure to test chemicals. Luminescence detection in the cell lysate after 48 ± 1 h of exposure at 37 ± 1.0 °C indicates luciferase induction and allows the discrimination between skin sensitisers and non-sensitisers.

TEST SUBSTANCE PREPARATION
A correction factor of 3.4 was used according to the purity/composition. Based on the results of a solubility test, the test item was dissolved in DMSO at 200 mM. In a 2-fold dilution series, 12 concentrations were prepared for application in cell culture medium. The final DMSO concentration in the cell culture medium was 1%.

CONCENTRATIONS:
0.98, 2.0, 3.9, 7.8, 16, 31, 63, 125, 250, 500, 1000 and 2000 µg/mL

CONTROLS:
VEHICLE CONTROL: Dimethyl sulfoxide (DMSO, Merck, Darmstadt, Germany)
Final concentration in the exposure medium: 1% (v/v)

POSITIVE CONTROL: Ethylene dimethacrylate glycol (EDMG, Sigma, Zwijndrecht, the Netherlands)
Solvent: DMSO, 1% (v/v) final concentration in the exposure medium
Concentration range: 7.8 – 250 µM

CELL CULTURE:
MEDIA:
Basic medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies, Bleiswijk, the Netherlands).
Maintenance medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 µg/mL).
Exposure medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

ENVIRONMENTAL CONDITIONS:
- Temperature (°C): 37 ± 1.0 (actual range: 36.0 – 37.2)
- CO2 (%): 5 ± 0.5
- Humidity (%): 80 – 100 (actual range: 29 – 93)

MAINTAINANCE:
Cells were sub-cultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock.

NUMBER OF REPLICATIONS: triplicates in two independent experiments

EXPERIMENTAL PROCEDURE:
Cells were seeded 10,000 cells / well in a 96-well format in basic medium. One plate was used for the luciferase activity measurements, and one parallel replicate was used for the MTT cell viability assay. After overnight cultivation, the medium was removed, replaced with antibiotic-free exposure medium and the test and control items were applied. The cells were exposed for 48 ± 1 h at 37 ± 1.0 °C. After the exposure period, luciferase activity was evaluated by luminescence measurement and cytotoxicity was assessed using the MTT viability assay.

LUCIFERASE ASSAY:
- Assay: Steady-Glo Luciferase Assay (Promega, Leiden, the Netherlands)
- Incubation time: 5 min at room temperature
- Luminometer: TECAN Infinite® M200 Pro Plate Reader

MTT VIABILITY ASSAY:
- MTT concentration: 5 mg/mL (11%)
- Incubation time: 3 – 4 h at 37 ± 1 °C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

EXPOSURE: 48 ± 1 h at 37 ± 1.0 °C

Results and discussion

Positive control results:

Ethylene dimethacrylate glycol (EDMG) was tested as positive control in a concentration range of 7.8 - 250 µM. In both experiments, luciferase activity increased dose-dependently with statistical significance. The induction at 250 µM was higher than 2-fold (2.51-fold and 2.79-fold in experiment 1 and 2, respectively). The EC1.5 was well within two standard deviations of the historical mean (61 µM in the first and 49 µM in the second experiment). Thus all acceptability criteria were met.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: 48 h exposure / Experiment 1

Parameter:

other: Imax

Value:

1.31

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Cytotoxicity was observed. The calculated IC30 was 159 µM and the calculated IC50 was 185 µM.

Results 2

Key result

Run / experiment:

other: 48 h exposure / Experiment 1

Parameter:

other: EC1.5

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Cytotoxicity was observed. The calculated IC30 was 159 µM and the calculated IC50 was 185 µM.

Results 3

Key result

Run / experiment:

other: 48 h exposure / Experiment 2

Parameter:

other: Imax

Value:

1.31

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Cytotoxicity was observed. The calculated IC30 was 108 µM and the calculated IC50 was 156 µM.

Results 4

Key result

Run / experiment:

other: 48 h exposure / Experiment 2

Parameter:

other: EC1.5

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Cytotoxicity was observed. The calculated IC30 was 108 µM and the calculated IC50 was 156 µM.

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Technical proficiency of the test system was demonstrated with 10 substances according to OECD 442D.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: Yes, the luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5-fold in at least one concentration. In addition, the EC1.5 of the positive control was within two standard deviations of the historical mean (61 µM and 49 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.51-fold and 2.79-fold in experiment 1 and 2, respectively).
- Acceptance criteria met for variability between replicate measurements: Yes, the average coefficient of variation of the luminescence reading for the vehicle control DMSO was below 20% (5.1% and 4.2% in experiment 1 and 2, respectively).

For details on historical control data of the positive control please refer to Table 4 under "Any other information in results incl. tables".

Any other information on results incl. tables

Table 1: Results of the KeratinoSens assay

	EC1.5 (µM)	Imax	IC30 (µM)	IC50 (µM)
Test item Experiment 1	NA	1.31	159	185
Test item Experiment 2	NA	1.31	108	156
Pos Control Experiment 1	61	2.51	NA	NA
Pos Control Experiment 2	49	2.79	NA	NA

Table 2: Luminescence and viability values of the first experiment

Concentration (µM)	Test item		Positive control
	Luminescence	Viability (%)	Luminescence	Viability (%)
0.98	0.93	113.4	-	-
2	0.97	100.8	-	-
3.9	1.06	90.3	-	-
7.8	1.03	95.6	0.92	120
16	1.06	88	1.03	95.5
31	1	95	1.22	108.1
63	0.91	91.2	1.51***	111.1
125	1.31	96	1.85***	114.6
250	0	-0.3	2.51***	111
500	0	-0.2	-	-
1000	0	-0.2	-	-
2000	0	-0.2	-	-

^***p<0.001 Student’s t test

Table 3: Luminescence and viability values of the second experiment

Concentration (µM)	Test item		Positive control
	Luminescence	Viability (%)	Luminescence	Viability (%)
0.98	1	108.7	-	-
2	0.99	98.6	-	-
3.9	0.97	84.9	-	-
7.8	0.98	79	1.06	106.8
16	1	81.3	1.09	95.4
31	0.96	74.2	1.31	91.3
63	0.98	78.5	1.63***	89
125	1.31	66.7	1.96***	83.6
250	0	-0.3	2.79***	80.3
500	-0.01	0.4	-	-
1000	-0.01	-0.2	-	-
2000	-0.01	-0.2	-	-

^***p<0.001 Student’s t test

Table 4: Historical control data for the positive control generated in the testing laboratory in June 2017 - June 2020

	Positive control
	EC1.5 (µM)	Imax
95% control Range	-3.8 – 123	-6.86 – 13.51
Mean	59.5	3.32
SD	31.6	5.09
n	500	500

SD = standard deviation; n = number of observations

Applicant's summary and conclusion

Interpretation of results:

other: no skin sensitising potential based on the key event “keratinocyte activation / inflammatory response”

Conclusions:

There is regulatory acceptance in the EU for the application of the KeratinoSens assay to address key event 2: activation of keratinocytes / inflammatory response in the skin sensitisation Adverse Outcome Pathway. Under the conditions of the test, the test substance was negative for keratinocyte activation in two independent experiments. The data generated with this method may not be sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of an integrated approach such as integrated approaches to testing and assessment (IATA).