N,N''-naphthalene-1,5-diylbis[N'-[3-[(2-ethylhexyl)oxy]propyl]urea]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant and in-house validated study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD TG no. 442D (adopted February 2015) - In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method

Principles of method if other than guideline:

Identification of keratinocyte activating substances with the transgenic keratinocyte cell line Lu-Sens derived from HaCaT cells. lt employs the reporter gene for luciferase under the control of an antioxidant response element and hence monitors Nrf-2 transcription factor activity. and is a "me-too" assay of the KeratinoSensTM assay.

GLP compliance:

yes (incl. QA statement)

Type of study:

other: in-vitro keratinocyte activation assay (LuSens)

Test material

Test material information

Constituent 1

Reference substance name:

N,N''-naphthalene-1,5-diylbis[N'-[3-[(2-ethylhexyl)oxy]propyl]urea]

EC Number:

275-276-0

EC Name:

N,N''-naphthalene-1,5-diylbis[N'-[3-[(2-ethylhexyl)oxy]propyl]urea]

Cas Number:

71216-01-8

Molecular formula:

C34H56N4O4

IUPAC Name:

N',N'''-naphthalene-1,5-diylbis(1-{3-[(2-ethylhexyl)oxy]propyl}urea)

Details on test material:

- Physical state: solid, white
-purity: 99.5%
- Purity test date: 2014
- Lot/batch No.: 130003P040
- Expiration date of the lot/batch: unlimited stability (lot manufactured in 2013)
- Stability under test conditions: stable
- Storage condition of test material: at room temperature
- Other: The test substance appeared to be homogeneous.

In vivo test system

Test animals

Species:

other: LuSens cells (derived from human keratinocyte cell line HaCaT)

Details on test animals and environmental conditions:

The reporter gene cell line LuSens was prepared in collaboration with the RWTH Aachen University. This keratinocyte cell line derived from HaCaT cells carries a reporter gene for luciferase under the control of an antioxidant-response-element (ARE) and hence monitors Nrf2 transcription factor activity. The sequence of the ARE promoter originates from the NADPH:quinone oxidoreductase1 gene from rats.


LuSens cells are routinely cultured in complete DMEM culture medium with high glucose supplemented with 10% fetal bovine serum (FBS),100 U/mL penicillin – 100 ug/mL streptomycin and 0.5 ug/mL puromycin in T75 culture flasks.

Study design: in vivo (non-LLNA)

Induction

Concentration / amount:

Stock solutions of the substances were prepared by dissolving in distilled water or in DMSO (final concentration of 200 mM) and diluted in 2-fold dilutions. Test substance solutions were further diluted in medium containing 1% FBS w/o puromycin to obtain a DMSO concentration of 1%.
The highest tested concentration in the main experiment was 1.22 fold of the concentration affording a viability of 75% (CV75). The additional concentrations were obtained by a 1:1exp2 dilution series of the CV75.

Cytotoxicity was determined via the MTT assay.

For substances dissolved in water, the final DMSO concentration was adjusted to 1%.

Challenge

Concentration / amount:

Stock solutions of the substances were prepared by dissolving in distilled water or in DMSO (final concentration of 200 mM) and diluted in 2-fold dilutions. Test substance solutions were further diluted in medium containing 1% FBS w/o puromycin to obtain a DMSO concentration of 1%.
The highest tested concentration in the main experiment was 1.22 fold of the concentration affording a viability of 75% (CV75). The additional concentrations were obtained by a 1:1exp2 dilution series of the CV75.

Cytotoxicity was determined via the MTT assay.

For substances dissolved in water, the final DMSO concentration was adjusted to 1%.

No. of animals per dose:

Two independent experiments were performed. In each experiment, three duplicates of each treatment were tested.

Details on study design:

LuSens cells from the working cell bank were thawed and cultured using culture containing antibiotics, under standard culture conditions for at least 2 weeks at passage >5 but not longer than 15 passages prior to testing.

Prior to substance incubation, cells were seeded in 96-well microtiter plates (0.12 mL of 0.83 x 10exp5 cells/ml cell suspensions), using culture medium without antibiotics for incubation for 24 hours.

Treatment was initiated by replacing regular cell culture medium with medium containing the test substance and a reduced content of FBS (1%).

Substance incubation was performed under standard cell culture conditions for 48 h and luciferase activity then determined using SteadyGlo™ (Promega, Germany) according to manufacturer’s instructions.

Positive control substance(s):

yes

Remarks:

Ethylene glycol dimethacrylate (0.015 mg/ml)

Results and discussion

Any other information on results incl. tables

Concentration (test substance) µg/mL	1st experiment fold induction             rel. viability [%]				2nd experiment fold induction             rel. viability [%]				3rd experiment fold induction             rel. viability [%]
	mean	SD	mean	SD	mean	SD	mean	SD	mean	SD	mean	SD
279 (Su)	1.08	0.12	82.9	3.2	1.10	0.19	87.6	7.0	0.96	0.24	76.7	5.2
335 (Su)	0.95	0.30	73.9	3.2	1.03	0.19	79.7	4.1	1.18	0.16	80.3	3.3
402 (Su)	1.01	0.46	72.1	6.6	0.94	0.36	80.9	5.7	0.99	0.23	77.7	4.9
482 (Su)	0.89	0.02	72.9	2.9	1.08	0.43	81.0	1.7	1.17	0.07	76.6	2.0
579 (Su)	1.67	0.25	75.3	3.7	1.30	0.31	76.6	3.4	0.99	0.43	75.2	8.1
694 (Su)	1.22	0.49	77.8	7.3	1.16	0.20	80.6	3.8	1.04	0.32	77.0	9.4
833 (Su)	1.86	0.39	71.3	6.5	0.83	0.15	79.5	2.9	1.20	0.04	74.4	2.4
1000 (Su)	1.20	0.13	84.6	0.6	1.03	0.45	83.1	0.6	1.20	0.15	80.1	5.8
VC	1.00	0.23	100.0	4.0	1.00	0.40	100.0	2.6	1.00	0.18	100.0	5.9
EGDMA (18 µg/mL)	5.39	1.14	100.8	5.3	6.52	1.12	103.8	2.6	5.30	0.44	107.0	1.4
LA (450 µg/mL)	1.24	0.21	116.7	1.5	1.23	0.32	119.2	2.4	1.04	0.27	113.1	2.8

Applicant's summary and conclusion

Interpretation of results:

other: no induction of antioxidant response genes in a keratinocyte cell line

Executive summary:

The keratinocyte activating potential of test substance was evaluated in the LuSens assay. For this purpose the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer. In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 9 concentrations of the test substance and cytotoxicity was determined thereafter by MTT assay. No decrease in cell viability below 75% was observed up to the maximum applicable concentration of 1000 μg/mL.

In the main test luciferase activity was measured after 48 hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 3 valid experiments were performed.

The test substance was a suspension in culture medium (2 x stock solutions) and in finally tested concentrations containing 1% DMSO. After 48 hours suspensions were noticed in all concentrations. after 48 hours of exposure to test substance luciferase activity in LuSens cells was not induced affording at least 70% viability in at least two consecutive concentrations of two independent experiments. From this it has to be concluded that test substance does not have a keratinocyte activating potential up to the maximum applicable concentration of 1000 μg/mL in the LuSens under the test conditions chosen.