2-(N-ethylanilino)ethanol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

February 04, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

2-(N-ethylanilino)ethanol

EC Number:

202-160-9

EC Name:

2-(N-ethylanilino)ethanol

Cas Number:

92-50-2

Molecular formula:

C10H15NO

IUPAC Name:

2-(N-Ethylanilino)ethanol

Test material form:

liquid - solid: mixture of

Details on test material:

At room temperature, the substance can be either a primrose yellow solid or a liquid.

Specific details on test material used for the study:

Batch No.: 18052904
Physical State: solid
Colour: primrose yellow
Retest Date: 28 May 2019

In vitro test system

Details on the study design:

The KeratinoSens™ assay is supposed to address the second key event of the skin sensitisation process as defined by the adverse outcome pathway (AOP), i.e. 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.
Two separate tests were carried out using the transgenic cell line KeratinoSens™ (Givaudan, Switzerland), a cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and were used for routine testing. Only cells at a low passage number <25 (P 11 in experiment 1; P 13 in experiment 2) were used.
Cells were cultured in 75 cm2 culture flasks (Greiner) in maintenance medium at 37  1°C and 5% CO2 in a humidified incubator. For test item exposure, cells were cultured in medium for test item exposure.
Dose Groups
1. Negative Control: 1% (v/v) DMSO in test item exposure medium
2. Positive Control: CA: 4 µM, 8 µM, 16 µM; 32 µM; 64 µM
3. Test Item: 12 concentrations of the test item
Experimental Procedure:
A cell suspension of 8 × 104 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1 × 104 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel, cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability.
After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 µL test item exposure medium. 50 µL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item.
All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.
Luciferase activity:
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 µL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light.
Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1 sec. before assessing the luciferase activity for 2 sec. This procedure was repeated for each individual well.
Cell viability:
For the cell viability plate the medium was replaced with 200 µL test item exposure medium. 27 µL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 200 µL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5% CO2 overnight (experiment 2) or over the weekend (experiment 1). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.
For each test item two independent repetitions using separately prepared test item solutions and independently harvested cells are necessary to derive a prediction. Each independent run consisted of three replicates for every concentration step of the test item and the positive control.
A KeratinoSens™ prediction is considered positive if the following conditions will be met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is <1000 µM
- an apparent overall dose-response for luciferase induction
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.

Results and discussion

Positive control results:

positive control concentration steps with significant luciferase activity induction >1.5 exceeded 1 for both experiments.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: Luciferase Activity

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: Luciferase Activity

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Any other information on results incl. tables

Table 1: 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	1.19	1.39	1.29	0.14
	8.00	1.25	1.20	1.23	0.03
	16.00	1.46	1.55	1.51	0.06
	32.00	2.05	2.09	2.07	0.03
	64.00	3.62	6.43	5.02	1.98
Test Item	0.98	0.95	1.22	1.08	0.19
	1.95	0.95	1.08	1.01	0.09
	3.91	0.95	1.06	1.01	0.08
	7.81	1.00	1.02	1.01	0.01
	15.63	1.01	1.05	1.03	0.03
	31.25	1.09	1.06	1.08	0.02
	62.50	1.13	1.19	1.16	0.04
	125.00	1.20	1.19	1.19	0.00
	250.00	1.27	1.43	1.35	0.12
	500.00	1.27	1.28	1.27	0.01
	1000.00	1.33	1.36	1.34	0.02
	2000.00	1.00	1.44	1.22	0.31

* = significant induction according to Student’s t-test, p<0.05

Table 2: Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Solvent Control	< 20%	8.5	pass	9.4	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.0	pass	3.0	pass
EC1.5 PC	± 2 x SD of historical mean	17.02	pass	14.84	pass
Induction PC at 64 µM	2.00 < x < 8.00	3.62	pass	6.43	pass

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the condition of this study the test item is considered as non sensitiser. The controls confirmed the validity of the study.

Executive summary:

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.

In the present study 2-(N-ETHYLANILINO)ETHANOL was dissolved in DMSO. Based on a molecular weight of 165.24 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 two separate experiments, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5value could be calculated.

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.