4-[2-[4-[benzylmethyl(ethyl)amino]phenyl]vinyl]-1-(2-hydroxyethyl)pyridinium acetate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 August 2017 to 13 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

4-[2-[4-[benzylmethyl(ethyl)amino]phenyl]vinyl]-1-(2-hydroxyethyl)pyridinium acetate

EC Number:

281-468-5

EC Name:

4-[2-[4-[benzylmethyl(ethyl)amino]phenyl]vinyl]-1-(2-hydroxyethyl)pyridinium acetate

Cas Number:

83950-14-5

Molecular formula:

C24H27N2O.C2H3O2

IUPAC Name:

4-[2-[4-[benzylmethyl(ethyl)amino]phenyl]vinyl]-1-(2-hydroxyethyl)pyridinium acetate

Test material form:

liquid

In vitro test system

Details on the study design:

Skin sensitisation (In vitro test system) - transgenic cell line KeratinoSens™ (Givaudan, Switzerland)
Based on a stock solution (40 mg/L arbitrary set at 200 mM) a set of twelve master solutions in 100% DMSO was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium (Dulbecco’s Modified Eagle 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 ± 1°C and 5% CO2. . After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

Results and discussion

Positive control results:

Cinnamic aldehyde (4 µM, 8 µM, 16 µM; 32 µM; 64 µM): Induction PC at 64 µM: 3.62 and 5.74 (within 2 times SD of historical control values)

In vitro / in chemico

Results

Results 1

Run / experiment:

other: 1

Parameter:

other: IC 1.5

Value:

11.53

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

IC 1.5 15.89 uM

Remarks on result:

positive indication of skin sensitisation

Remarks:

cell viability at lowest concentration inducing > 1.5 fold increase 109.4%

Results 2

Run / experiment:

other: 2

Parameter:

other: IC 1.5

Value:

9.56

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

IC 1.5 10.36 uM

Remarks on result:

positive indication of skin sensitisation

Remarks:

cell viability at lowest concentration inducing > 1.5 fold increase 105.9%

Any other information on results incl. tables

Induction of Luciferase Activity – Overall Induction

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	1.19	1.18	1.18	0.01
	8.00	1.37	1.38	1.37	0.00
	16.00	1.50	1.79	1.65	0.21	*
	32.00	2.11	2.54	2.33	0.31	*
	64.00	3.62	5.74	4.68	1.50	*
Test Item	0.98	1.06	1.14	1.10	0.06
	1.95	0.98	1.01	1.00	0.02
	3.91	0.87	0.89	0.88	0.02
	7.81	0.95	1.21	1.08	0.18
	15.63	2.11	2.52	2.31	0.29	*
	31.25	4.82	5.83	5.33	0.72	*
	62.50	9.65	11.09	10.37	1.02	*
	125.00	16.82	10.18	13.50	4.70
	250.00	0.01	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

Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Solvent Control	< 20%	6.4	pass	8.2	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	15.89	pass	10.36	pass
Induction PC at 64 µM	2.00 < x < 8.00	3.62	pass	5.74	pass

 

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Based on the in vitro KeratinoSens™ assay the substance is considered sensitizing to the skin.

Executive summary:

In an in vitro KeratinoSens™ assay the sensitising potential of the substance by addressing the second molecular key event of the adverse outcome pathway (AOP) for skin sensitization, namely activation of keratinocytes, 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 (I_max) induction of 16.82 was determined at a substance concentration of 125 µM. The corresponding cell viability was 6.6%. The lowest tested concentration with a significant luciferase induction >1.5 (2.11) was found to be 15.63 µM. The corresponding cell viability was >70% (109.4%).The calculated EC_1.5 was < 1000 µM (11.53 µM).

In the second experiment, a max luciferase activity (I_max) induction of 11.09 was determined at a substance concentration of 62.5 µM. The corresponding cell viability was 45.8%. The lowest tested concentration with a significant luciferase induction >1.5 (2.52) was found to be 15.63 µM. The corresponding cell viability was >70% (105.9%).The calculated EC_1.5 was < 1000 µM (9.56 µM).

A dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

The result is an indication for skin sensitizing properties of the substance. However, as this involves only the second key event in the AOP for skin sensitization, no definitive conclusion can be drawn.