2-(oxolan-2-ylmethoxy)ethanol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

20 Oct - 10 Nov 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

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)

Version / remarks:

adopted in 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

2-[(oxolan-2-yl)methoxy]ethan-1-ol

EC Number:

608-659-1

Cas Number:

31692-85-0

Molecular formula:

C5H10O2[C2H4O]n, n = 0, 1, 2, 3, 4, ... (data given for max n of 4)

IUPAC Name:

2-[(oxolan-2-yl)methoxy]ethan-1-ol

In vitro test system

Details on the study design:

TEST METHOD
The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test substance 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 sensitiser and non-sensitisers.

TEST CELL LINE
- Type: KeratinoSens™
- Source: Givaudan, Switzerland
- Passage number: 8 (experiment 1), 12 (experiment 2)

CELL CULTURE CONDITIONS
- Type and identity of media:
Maintenance medium: Dulbecco’s Modified Eagle Medium (GlutaMAX TM) supplemented with 1.0 g/L D-glucose and Na-pyruvate, 10% fetal bovine calf serum and 1% geneticin (final concentration 500 μg/mL)
Assay medium: Dulbecco’s Modified Eagle Medium (GlutaMAX TM) supplemented with 1.0 g/L D-glucose and Na-pyruvate and 10% fetal bovine calf serum
Test substance exposure medium: Dulbecco’s Modified Eagle Medium (GlutaMAX TM) supplemented with 1.0 g/L D-glucose and Na-pyruvate and 1% fetal bovine calf serum
- Temperature (°C): 37 ± 1.0
- CO2 (%): 5.0

TEST CONCENTRATIONS
0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM

CONTROLS
Vehicle control
- Substance: dimethyl sulfoxide (DMSO)
- Final concentration: 1%, in test substance exposure medium

Positive control
- Substance: cinnamic aldehyde
- Final concentration: 4 - 64 μM, in 1% DMSO

EXPOSURE CONDITIONS
- Exposure duration: 48 ± 1 h

NUMBER OF REPLICATIONS: two independent exp. with 3 replicates

DETERMINATION OF CELL VIABILITY
- Method: MTT assay
- MTT concentration: 5 mg/mL (stock solution) in DPBS
- Incubation time: 4 h at 37 ± 1°C
- Device: plate reader
- Wavelength: 600 nm

DETERMINATION OF LUMINESCENCE
- Luciferase reagent: Luciferase Assay Kit (Promega, Cat. no. E1531, Lot No. 0000246522)
- Device: plate reader

Results and discussion

Positive control results:

The positive control induced a significant increase in the luciferase activity at a concentration of 32 µM (2.09 and 2.03 in experiment 1 and 2, respectively) and 64 µM (3.95 and 3.24 in experiment 1 and 2, respectively). The EC 1.5 value of the positive control was found to be 16.09 and 17.22 µM in experiment 1 and 2, respectively.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: experiment 1, test substance

Parameter:

other: maximum luciferase activity induction (mean of 3 replicates)

Value:

1.35

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: at 2000 µM

Results 2

Key result

Run / experiment:

other: experiment 2, test substance

Parameter:

other: maximum luciferase activity induction (mean of 3 replicates)

Value:

1.21

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: at 2000 μM

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY: not stated in the report

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
The average coefficient of variation of the luminescence reading for the vehicle control DMSO was 7.8% in experiment 1 and 5.2% in experiment 2 and thus < 20%

- Acceptance criteria met for positive control: yes
The luciferase activity induced by the positive control at a concentration of 64 µM was 3.95 in experiment 1 and 3.24 in experiment 2 (range of acceptance criteria: 2 - 8).
The EC 1.5 value of the positive control was 16.09 µM in experiment 1 and 17.22 µM in experiment 2 and thus within two standard deviations of the historical mean of the test facility (7 - 34 µM)

Any other information on results incl. tables

Table 2: Results of the cytotoxicity measurement

	Concentration [μM]	Cell viability (%)
		Exp. 1	Exp. 2	Mean ± SD
Vehicle control	-	100.0	100.0	100 ± 0.0
Positive control	4.00	100.2	100.1	100.2 ± 0.1
	8.00	112.2	103.5	107.9 ± 6.1
	16.00	124.3	110.4	117.3 ± 9.8
	32.00	129.6	113.7	121.7 ± 11.3
	64.00	135.1	117.1	126.1 ± 12.8
Test substance	0.98	87.7	96.9	92.3 ± 6.5
	1.95	81.3	99.9	90.6 ± 13.2
	3.91	80.0	97.4	88.7 ± 12.3
	7.81	90.1	97.9	94.0 ± 5.5
	15.63	87.7	95.5	91.6 ± 5.5
	31.25	91.7	97.6	94.6 ± 4.2
	62.50	94.2	97.9	96.1 ± 2.6
	125.00	102.3	95.4	98.9 ± 4.9
	250.00	109.3	98.9	104.1 ± 7.4
	500.00	113.0	102.5	107.8 ± 7.4
	1000.00	115.2	110.0	112.6 ± 3.6
	2000.00	146.0	114.2	130.1 ± 22.4

Table 3: Overall induction of luciferase activity

	Concentration [μM]	Fold induction
		Exp. 1				Exp. 2
		1	2	3	Mean ± SD	1	2	3	Mean ± SD
Vehicle control	-	1.00	1.00	1.00	1.00 ± 0.1	1.00	1.00	1.00	1.00 ± 0.0
Positive control	4.00	1.12	1.18	1.22	1.17 ± 0.05	1.05	1.18	1.10	1.11 ± 0.06
	8.00	1.31	1.29	1.18	1.26 ± 0.07	1.26	1.31	1.41	1.33 ± 0.08
	16.00	1.47	1.55	1.47	1.50 ± 0.04	1.43	1.45	1.49	1.46 ± 0.03
	32.00	2.12	1.98	2.17	2.09 ± 0.10 *	1.88	2.04	2.18	2.03 ± 0.15 *
	64.00	3.92	3.82	4.10	3.95 ± 0.14 *	3.14	3.12	3.47	3.24 ± 0.19 *
Test substance	0.98	0.92	0.93	0.90	0.92 ± 0.02	1.04	1.01	0.98	1.01 ± 0.03
	1.95	0.98	0.87	0.94	0.93 ± 0.05	1.04	1.04	1.01	1.03 ± 0.02
	3.91	1.02	1.02	0.94	1.00 ± 0.05	1.03	1.08	1.09	1.07 ± 0.04
	7.81	1.12	0.97	0.98	1.02 ± 0.09	0.99	1.01	1.24	1.08 ± 0.14
	15.63	1.04	1.00	0.95	1.00 ± 0.05	1.06	1.04	1.06	1.05 ± 0.01
	31.25	1.06	1.12	0.99	1.05 ± 0.07	1.03	1.04	1.07	1.05 ± 0.02
	62.50	1.07	1.06	1.04	1.06 ± 0.02	0.98	1.05	1.06	1.03 ± 0.04
	125.00	1.10	1.06	1.05	1.07 ± 0.03	1.11	1.06	1.12	1.10 ± 0.03
	250.00	1.04	1.03	0.99	1.02 ± 0.03	1.13	1.10	1.15	1.13 ± 0.03
	500.00	1.25	1.08	1.06	1.13 ± 0.11	1.05	1.21	1.12	1.13 ± 0.08
	1000.00	1.02	1.03	0.96	1.00 ± 0.04	1.14	1.25	1.19	1.20 ± 0.05
	2000.00	1.56	1.36	1.14	1.35 ± 0.21	1.23	1.23	1.18	1.21 ± 0.03

*: significant induction according to Student’s T-test, p<0.05

Applicant's summary and conclusion

Interpretation of results:

other: no skin sensitising potential based on the key event “inflammatory response in keratinocytes”

Conclusions:

Under the conditions of the test, the test substance did not have a keratinocyte activating potential. The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.