Reaction products of diazotized Dodecyl Aniline coupled with 8-acetylamine-1-hydroxynaphthalen-3,6-disulphonic Acid, sodium salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from 29. Apr. 2019 to 16. May. 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2018

Deviations:

yes

Remarks:

see "principles of method if other than guideline"

Principles of method if other than guideline:

- Negative control (DL-Lactic Acid) has a purity (90.5 %) that is lower than the one suggested in the OECD Guideline 442D. This difference does not change the results and course of the study and has no impact on the results since the water and residual content is considered so that during the requested treatment 5000 µM of the pure DL-Lactic acid is tested.
-Positive control (EGDMA) has a purity (98 %) that is lower than the one suggested in the OECD Guideline 442D. This difference does not change the results and course of the study and has no impact on the results since the water and residual content is considered so that during treatment 120 µM of the pure EGDMA is tested.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

disodium 3-[(1E)-2-(4-dodecylphenyl)diazen-1-yl]-5-acetamido-4-hydroxynaphthalene-2,7-disulfonate

EC Number:

946-819-5

Molecular formula:

Not applicable for UVCB substance

IUPAC Name:

disodium 3-[(1E)-2-(4-dodecylphenyl)diazen-1-yl]-5-acetamido-4-hydroxynaphthalene-2,7-disulfonate

Test material form:

solid: particulate/powder

In vitro test system

Details on the study design:

PREPARATION OF TEST ITEM
-Preparation:
The solubility of the test item was determined in a non-GLP pre-test in dimethyl sulfoxide (DMSO) and medium (Dulbecco´s Modified Eagle Medium, DMEM). The test item was insoluble in DMSO and medium at the required concentration (200 mg/mL) after 30 min sonication. The test item is insoluble at the concentration 100 mg/mL in medium, but solu-ble in DMSO at this concentration after 30 min sonication. Therefore, DMSO was used as solvent.
The highest test item concentration in the Cytotoxicity Range Finder Test (CRFT) was 1000 µg/mL. Since the final concentration of the solvent during treatment is limited to 1 %, a stock solution containing 100 mg/mL (CRFT) and 62.5 mg/mL (experiment) test item in DMSO was prepared and sonicated for 30 min. Subsequent dilution to 1 % finally yielded a maximum concentration of 1000 µg/mL in the CRFT and 62.5 µg/mL in the experiment.
For that, the stock solution was first used to prepare a geometric series of solutions (CRFT: factor 2; main experiment: factor 1.2) on a master plate. Afterwards all concentrations were further diluted (1:25) in medium no. 3 on a dilution plate. Another 1:4 dilution was achieved by adding 50 µL of each concentration of the dilution plate to the corresponding wells of the test plate containing the cells as well as 150 µL medium no. 3. In the end, the total dilution factor was 1:100. The stock solution as well as the dilutions were freshly prepared on the day of treatment.

TEST SYSTEM
-Reasons for the Choice of the LuSens Cell Line
The LuSens cell line was specially designed for this test system by the BASF SE (Ludwig-shafen, Germany). It employs the use of a luciferase reporter gene placed under the control of the antioxidant response element (ARE) and hence monitors Nrf-2 transcription factor activity. For designing this cell line, a human keratinocyte cell line (provided by RWTH, Aachen, Germany) was transfected with the pGL4.20 [luc2/Puro] vector (Promega, Germany) carrying the regulatory antioxidant response element (ARE) upstream of the luciferase gene (Luc2, Promega, Germany) at the Institute of Anatomy and Cell Biology of the RWTH, Aachen (laboratory of PD Dr. Wruck).

-Cell Cultures:
The LuSens cell line was obtained from the BASF SE (Ludwigshafen, Germany). For mycoplasma contamination screened stocks of LuSens cells are stored in liquid nitrogen in the cell bank of the lab to allow a continuous stock of cells (mycoplasma contamination free), which guarantees similar parameters of the experiment and reproducible char-acteristics of the cells.
For the Cytotoxicity Range Finder Assay cells of passage 14 were used. For the repetitions, cells of passage 6 (repetition I) and 8 (repetition II) were used. After thawing the cells were cultivated in DMEM (9 % FCS (Fetal calf serum)) in cell culture flasks at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.

DEMONSTRATION OF PROFICIENCY
Prior to routine use, the validity of the lab was demonstrated in a proficiency study. 22 proficiency chemicals indicated by the OECD 442 D were tested. The 10 proficiency chemicals which are indicated by the current version of the OECD 442D were all included in the proficiency studyand were categorized correctly regarding the expected LuSens prediction. The reference range (EC1.5, CV75) was correctly obtained for 8 substances. From 22 proficiency chemicals more than 80 % (95 %) of the results were correctly categorized. Therefore, the proficiency of the LuSens test was demonstrated. For all control substances historical data are available (will be given in the final report), which demonstrates the reliability and the validity of those substances.

PERFORMANCE OF THE STUDY
-Cytotoxicity Range Finder Test:
A Cytotoxicity Range Finder Test (CRFT) was performed in order to determine the concentration range applicable for the repetitions. In the CRFT cytotoxicity was determined by measuring the cell viability with MTT. This yellow tetrazole is reduced to purple formazan in viable cells and can therefore be used for assessing the cell metabolic activity and therefore the cell viability. A reduction of the viability below 70 % is defined as a cytotoxic effect.
In the CRFT the following 12 nominal concentrations of the test item were tested:
0.49 µg/mL, 0.98 µg/mL, 1.95 µg/mL, 3.91 µg/mL, 7.81 µg/mL, 15.63 µg/mL, 31.25 µg/mL, 62.5 µg/mL, 125 µg/mL, 250 µg/mL, 500 µg/mL, 1000 µg/mL.
At the time of seeding the cells were 80 % confluent. The cells were washed twice with PBS (without Ca2+/Mg2+) containing 0.05 % EDTA. Afterwards the cells were trypsinized (by adding Trypsin/EDTA to the flask) until the cells detached. To stop this reaction, me-dium no. 2 was added. After centrifugation (5 min at 380 * g), the supernatant was dis-carded and the cells were resuspended in medium no. 2. After quantification by cell counter, the cell suspension was adjusted to 83 000 (± 10 %) cells per mL. 120 µL of the cell suspension (≙ 10 000 cells) were seeded in a clear flat bottom 96 well plate. The plate was incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere for 24 h 5 min.
After the incubation time the medium was removed from the cells and 150 µL medium no. 3 was added to each well. Afterwards, 50 µL of the single test item concentrations as well as controls were added to the cells in triplicates (only test item concentrations). Twelve wells were used as solvent control, 6 wells were used as growth control (cells + medium no. 3), 3 wells were used as negative control, 2 wells were used as positive control and 1 well was used as blank control. The plate was sealed with breathable tapes to avoid evap-oration of volatile compounds and to avoid cross contamination between wells. Afterwards, the plate was incubated for 48 h at 37 ± 1 °C in a humidified atmosphere containing 5.0 ± 0.5 % CO2.
For the viability assay the MTT working solution was prepared by mixing 9 parts of medium no. 3 with 1 part of MTT solution. All solutions were removed from the wells of the 96 well plate and 200 µL MTT working solution was added to each well. The plate was incu-bated for 2 h at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere. Afterwards, the solution was removed and 100 µL MTT-lysis buffer was added to each well. The plate was agitated for 5 min before it was measured at a wavelength of 570 nm and of 690 nm at the photometer.
For calculation of the relative viability a validated Microsoft Excel® file was used.

-Dose Selection for Experiment I and II:
In accordance with the OECD guideline 442D, the maximum final test item concentration should be 2000 µM. For a test chemical which has no defined molecular weight, the final test item concentration 2000 µg/mL can also be used. Alternative concentrations may be used upon justification (e.g. in case of cytotoxicity or poor solubility).
In the case of a cytotoxic result, the concentrations for the experiment should be determined so that at least one of them is in the cytotoxic range.
Since a cytotoxic reaction was observed in the CRFT, the highest “calculated” concentration for the experiments would be 19.3 µg/mL (1.2*CV75). The aim was to have one cytotoxic and three non-cytotoxic concentrations, so to be save to have at least one cytotoxic concentration, we started the experiments with the concentration of 62.5 µg/mL. 12 concentrations were analysed to cover a range as huge as possible, since the dilutions steps are just 1.2.
The following 12 nominal concentrations were chosen for repetition I and II:
8.4 µg/mL, 10.1 µg/mL, 12.1 µg/mL, 14.5 µg/mL, 17.4 µg/mL, 20.9 µg/mL, 25.1 µg/mL, 30.1 µg/mL, 36.2 µg/mL, 43.4 µg/mL, 52.1 µg/mL, 62.5 µg/mL
A test item concentration inducing a viability reduction below 70 % is considered as cytotoxic and is not allowed to be evaluated for luciferase induction.

- Experimental Parameters of Experiment I and II
Repetition I and II were performed in the same way. At the time of seeding the cells were 80 % confluent. The cells were washed twice with PBS (without Ca2+/Mg2+) containing 0.05% EDTA. Afterwards the cells were trypsinized until the cells detached. To stop this reaction, medium no. 2 was added. After centrifuga-tion (5 min at 380 * g), the supernatant was discarded and the cells were resuspended in medium no. 2. After quantification, the cell suspension was adjusted to 83 000 (±10 %) cells per mL. 120 µL of the cell suspension (≙ 10 000 cells) were seeded in two clear flat bottom 96 well plates (one for viability and one for luciferase induction measurement). Both plates were incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere for 24 h in repetition I and 24 h 10 min in repetition II.
The treatment procedure was performed on both 96 well plates identically:
After the incubation time the medium was removed from the cells and 150 µL medium no. 3 were added to each well. Afterwards 50 µL of each single test item concentration and the controls were added to the cells in triplicates (test item concentrations). 24 wells were used for solvent control, 12 wells were used for growth control (cells + medium no. 3), 6 wells were used for negative control, 5 wells for positive control and 1 well for blank. The plates were sealed with breathable tape to avoid evaporation of volatile compounds and to avoid cross contamination between wells. Afterwards the plates were incubated for 48 h at 37 ± 1 °C in a humidified atmosphere containing 5.0 ± 0.5 % CO2.
For the evaluation of the viability, one of the plates was used:
The MTT working solution was prepared by mixing 9 parts of medium no. 3 with 1 part of MTT solution. All solutions were removed from the wells of the 96 well plate and 200 µL MTT working solution were added to each well. The plate was incubated for 2 h at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere. Afterwards the solution was re-moved and 100 µL of lysis buffer were added to each well. The plate was agitated for 5 min before it was measured at 570 nm and at 690 nm (reference) at the photometer. The cell viability is measured by the reduction of the tetrazolium dye MTT (3-(4,5-Dimethyl thiazole 2-yl)-2,5-diphenyltetrazolium-bromide) (yellow colour) to its insoluble formazan (purple colour) in living cells and therefore indicates the amount of living cells. After the measurement of the colour change, the values were transferred in a validated spreadsheet for the calculation of the viability.
For the evaluation of the Luciferase induction, the second plate was used:
For the evaluation of the Luciferase expression, after the 48 h exposure period, all solutions were removed from the wells and the cells were washed twice with 300 µL PBS (with Ca2+/Mg2+). Afterwards 100 µL per well of a Lysis buffer were added to the cells and incubated for 5 min at room temperature. During this process, the plate was slightly moved. Afterwards 100 µL Steady-Glo® Reagent were added to each well and the plate was shaken again slowly for 5 min at room temperature. Then, 160 µL per well were trans-ferred to a white flat bottom 96 well plate and the luminescence was measured for 2 sec-onds using a luminometer.
For calculation of the luciferase induction as well as the relative viability a validated Microsoft Excel® file was used.

DATA EVALUATION
- Calculation of Relative Viability
The calculation of the relative Viability [%] was performed as follows:
All wells were corrected = OD570 value - OD690 value
For the following calculation only the corrected values were used.
Viability = [( Vsample - Vblank) / (Vsolvent - Vblank)] * 100

Vsample= MTT-absorbance reading in the test chemical well
Vblank= MTT-absorbance reading in the blank well containing no cells and no treatment
Vsolvent= is the average MTT-absorbance reading in wells containing cells and solvent
Afterwards the mean value of the single replicates was calculated.

- Calculation of CV75
The CV75-value (relative survival rate) was calculated by linear interpolation. This value is the substance concentration at which cell viability is 75% compared to the control.
The CV75 was calculated as follows:
CV75 = (Cb - Ca) * [(75 - Vb) / (Vb - Va)] + Cb

Ca = lowest concentration in µM or µg/mL with > 75 % cell viability
Cb = highest concentration in µM or µg/mL with < 75 % reduction in viability
Va = % viability at the lowest concentration with > 75 % cell viability
Vb = % viability at the highest concentration with < 75 % cell viability

- Calculation of Luciferase fold induction
Fold induction = [(Lsample - Lblank) / (Lsolvent - Lblank)]

Lsample = luminescence reading (RLU) in the test chemical well
Lblank = luminescence reading in blank well containing no cells and no treatment
Lsolvent = average luminescence reading in wells containing cells and solvent

Afterwards the mean value of the single replicates was calculated.

Results and discussion

In vitro / in chemico

Results

Run / experiment:

other: Experiment I and II, test item concentrations (8.4, 10.1, 12.1, 14.5, 17.4, 20.9 and 25.1 μg/ml)

Parameter:

other: luciferase induction

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

- Cytotoxicity Range Finder Test
No cytotoxic effect was observed at the controls as well as the test item concentrations 0.49 µg/mL to 15.36 µg/mL. The viability values were all above 74 %. In all other test item concentrations (31.25 µg/mL to 1000 µg/mL) cytotoxic effects were observed with relative viability values below 65 %.
No precipitates were observed in the treatments.
The CRFT was valid and could be used for the determination of the concentrations to be tested in the experiment.
The CV75 value was also calculated and is 16.1 µg/mL.

- Experiment I
All control substances indicated the expected effect. No considerable reduction of the viability was detected (all values ≥ 85 %). Regarding the Luciferase induction, the growth control and the negative control did not exceed the threshold of 1.5 fold in comparison to the solvent control (growth control: 0.9 fold, negative control: 1.0 fold). However, the positive control induced a clear effect with an induction value of 7.8 fold in comparison to the solvent control.
No cytotoxic effect was observed at the test item concentrations 8.4 µg/mL to 25.1 µg/mL. The viability values were all > 70 % and therefore analysable for luciferase induction. Cytotoxic effects were observed at the concentrations 30.1 µg/mL to 62.5 µg/mL. Due to cytotoxicity these values were not used for the final evaluation of luciferase induction.
In the Luciferase assay, none of the tested non cytotoxic concentrations induced a luciferase induction above or equal 1.5 fold in comparison to the solvent control.

- Experiment II
All control substances indicated the expected effect. No considerable reduction of the vi-ability was detected (all values ≥ 88 %). Regarding the Luciferase induction, the growth control and the negative control did not exceed the threshold of 1.5 fold in comparison to the solvent control (growth control: 0.9 fold, negative control: 1.1 fold). However, the posi-tive control induced a clear effect with an induction value of 7.3 fold in comparison to the solvent control.
No cytotoxic effect was observed at the test item concentrations 8.4 µg/mL to 25.1 µg/mL. The viability values were all ≥ 74 % and therefore analysable for luciferase induction. Cytotoxic effects were observed at the concentrations 30.1 µg/mL to 62.5 µg/mL. Due to cytotoxicity these values were not used for the final evaluation of luciferase induction.
In the Luciferase assay, none of the tested non cytotoxic concentrations induced a luciferase induction above or equal 1.5 fold in comparison to the solvent control.

VALIDITY CRITERIA:
- The average induction for the positive control should be ≥ 2.5 fold and it should have a relative viability of at least 70 %.
- The induction triggered by the negative control and growth control should be < 1.5 fold as compared to the induction of the solvent control and the viability should be above 70 %.
- The average percentage standard deviation (luciferase induction) of the variability in at least 21 solvent control wells should be below 20 %.
- At least 3 test concentrations must be within viability limits, i.e. have relative viability of at least 70 %.
- In case a result is to be considered negative, at least one concentration should be cytotoxic, i.e. have a cell viability < 70 %, or the maximum concentration of 2000 µM (2000 µg/mL) should have been tested.

All validity criteria about experiment I and II were met. Therefore, the study is valid.
Experiment I
-Positive control: Fold induction: 67.8; Relative viability: 85.8 %
-Negative control: Fold induction: 1.0; Relative viability: 108.2 %
-Growth control: Fold induction: 0.9; Relative viability: 132.1 %
-Average percentage standard deviation: 7.66 %
-7 concentrations are analysable
-5 concentrations cytotoxic (negative result)
Experiment II
-Positive control; Fold induction: 7.3; Relative viability: 88.3 %
-Negative control: Fold induction: 1.1; Relative viability: 108.5 %
-Growth control: Fold induction: 0.9; Relative viability: 136.6 %
-Average percentage standard deviation: 8.02 %
-7 concentrations are analysable
-5 concentrations cytotoxic (negative result)

CLASSIFICATION
Each valid experiment (i.e. meeting all acceptance criteria, according to the procedure described above) is interpreted as follows:
A test compound is considered to have the potential to activate the Nrf2 transcription factor if the luciferase induction is ≥ 1.5 fold and statistically significant compared to the vehicle control in 2 (or more than) consecutive non-cytotoxic (relative viability ≥ 70 %) tested concentrations whereby at least three tested concentrations must be non-cytotoxic in two independent valid repetitions.
A test compound is considered not to have the potential to activate the Nrf2 transcription factor if the effects mentioned above are not observed.
A negative result obtained with test chemicals that do not form a stable dispersion and/or were not tested up to 2000 µM (or 2000 µg/mL for test chemicals with no defined molecular weight) and for which no cytotoxicity is observed in any of the tested concentration should be considered as inconclusive.
A minimum of two valid and independent repetitions need to indicate a positive or negative result. If the first two repetitions come to the same result (i.e. either being negative or being positive) no further testing is required. In case that the first two repetitions give discordant results (i.e. one is negative and the other is positive), a third independent repetition needs to be conducted to complete the study. The skin sensitizing potential (corresponding to the potential to activate the Nrf2 transcription factor) of a test substance is determined by the result of the majority of the repetitions of an experiment. If two of two or two of three repetitions are negative/positive, the substance is considered as negative/positive.

The luciferase induction was not above or equal to 1.5 fold in comparison to the solvent control in 2 (or more than) consecutive non-cytotoxic test item concentrations in repetition I and II.

Therefore, the test item is considered not to have the potential to activate the Nrf2 transcription factor under the conditions of the LuSens test.

Any other information on results incl. tables

Results of Relative Viability [%] in CRFT

Parameter	Concentration	Relative	Standard	Standard
		Viability	Deviation	Deviation
	[µg/mL]	[%]		[%]
Solvent Control	-	100.0	3.97	3.97
Growth Control	-	144.6	3.76	2.60
Negative Control	5000 µM	106.5	2.92	2.75
Positive Control	120 µM	95.6	1.76	1.84
Test item	0.49	98.5	1.91	1.94
Test item	0.98	93.9	1.34	1.43
Test item	1.95	90.0	1.35	1.51
Test item	3.91	82.0	2.96	3.61
Test item	7.81	74.7	3.17	4.25
Test item	15.63	75.3	2.10	2.79
Test item	31.25	64.9	2.61	4.02
Test item	62.5	15.8	2.79	17.66
Test item	125	2.4*	0.09	3.77
Test item	250	3.0*	0.24	7.90
Test item	500	5.4*	0.31	5.71
Test item	1000	17.4*	2.03	11.68

*=In these concentrations reddish staining was visible in the wells.

Results of Experiment I

		Induction of Luciferase			Viability of the Cells
Parameter	Concentration	Induction	Standard	Standard	Relative	Standard	Standard
			Deviation	Deviation	Viability	Deviation	Deviation
	[µg/mL]	fold		[%]	[%]		[%]
Solvent Control	-	1.0	0.08	7.66	100.0	3.78	3.78
Growth Control	-	0.9	0.07	8.33	132.1	3.29	2.49
Negative Control	5000 µM	1.0	0.07	7.22	108.2	1.64	1.51
Positive Control	120 µM	7.8	0.47	6.06	85.8	3.05	3.55
Test item	8.4	0.6	0.05	9.07	82.3	0.48	0.58
Test item	10.1	0.6	0.04	7.12	79.4	1.33	1.67
Test item	12.1	0.6	0.04	7.13	75.0	3.36	4.48
Test item	14.5	0.6	0.04	7.35	72.0	2.31	3.20
Test item	17.4	0.6	0.01	2.15	70.8	1.91	2.70
Test item	20.9	0.6	0.05	8.07	73.1	4.92	6.73
Test item	25.1	0.6	0.05	7.82	72.2	3.51	4.87
Test item	30.1	0.6*	0.03	5.45	67.9	6.44	9.47
Test item	36.2	0.6*	0.06	10.72	66.0	7.07	10.72
Test item	43.4	0.5*	0.03	5.21	58.2	5.80	9.97
Test item	52.1	0.4*	0.05	11.71	38.9	7.85	20.19
Test item	62.5	0.3*	0.02	7.29	28.5	10.52	36.98

* = Due to cytotoxicity, these values were not used for the final evaluation of luciferase induction.

Results of Experiment II

		Induction of Luciferase			Viability of the Cells
Parameter	Concentration	Induction	Standard	Standard	Relative	Standard	Standard
			Deviation	Deviation	Viability	Deviation	Deviation
	[µg/mL]	fold		[%]	[%]		[%]
Solvent Control	-	1.0	0.08	8.02	100.0	2.44	2.44
Growth Control	-	0.9	0.06	6.27	136.6	4.49	3.29
Negative Control	5000 µM	1.1	0.03	2.91	108.5	4.45	4.10
Positive Control	120 µM	7.3	0.26	3.55	88.3	3.53	3.99
Test item	8.4	0.6	0.02	3.84	81.2	1.74	2.14
Test item	10.1	0.6	0.05	7.80	76.1	0.91	1.20
Test item	12.1	0.6	0.02	3.90	78.5	1.93	2.46
Test item	14.5	0.6	0.06	10.97	79.7	2.06	2.58
Test item	17.4	0.6	0.04	6.77	76.5	5.58	7.29
Test item	20.9	0.6	0.01	0.81	74.8	2.33	3.11
Test item	25.1	0.6	0.08	12.97	74.2	5.46	7.36
Test item	30.1	0.6*	0.05	7.61	69.2	3.15	4.55
Test item	36.2	0.5*	0.03	6.35	65.0	2.01	3.09
Test item	43.4	0.6*	0.03	5.33	55.7	1.97	3.55
Test item	52.1	0.5*	0.01	0.96	45.3	2.37	5.24
Test item	62.5	0.4*	0.00	1.03	29.3	2.86	9.79

* = Due to cytotoxicity, these values were not used for the final evaluation of luciferase induction.

Historical Data of the Negative Control and the Positive Control

	Σ	Mean induction value	Standard	Range
			deviation
Positive Control	214	5.4	1.475	2.1 – 14.7
(EGDMA)
Negative control	262	1.0	0.085	0.7 – 1.3
(DL-Lactic acid)

Nominal and Real Test Item Concentrations

Cytotoxicity Range Finder Tests (CRFT)		Repetition I		Repetition II
nominal concentrations [µg/mL]	real concentrations	nominal concentrations [µg/mL]	real concentrations	nominal concentrations [µg/mL]	real concentrations
	[µg/mL]		[µg/mL]		[µg/mL]
0.49	0.49	8.4	8.5	8.4	8.5
0.98	0.98	10.1	10.1	10.1	10.2
1.95	1.95	12.1	12.2	12.1	12.3
3.91	3.91	14.5	14.6	14.5	14.7
7.81	7.81	17.4	17.5	17.4	17.7
15.63	15.63	20.9	21.0	20.9	21.2
31.25	31.26	25.1	25.2	25.1	25.5
62.5	62.5	30.1	30.3	30.1	30.6
125	125	36.2	36.3	36.2	36.7
250	250	43.4	43.6	43.4	44.0
500	500	52.1	52.3	52.1	52.8
1000	1000	62.5	62.8	62.5	63.4

Applicant's summary and conclusion

Interpretation of results:

other: no potential to activate the Nrf2 transcription factor

Conclusions:

The test item was negative in the LuSens assay under the experimental conditions of this study.

Executive summary:

This in vitro study evaluates the potential of the test item to activate the Nrf2 transcription factor by using the LuSens cell line, according to the OECD guideline 442D (2018). This test is part of a tiered strategy for the evaluation of skin sensitization potential. Therefore, data generated should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

The assay included a cytotoxicity range finder test (CRFT) and one experiment, consisting of two independent repetitions (repetition I and II) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test item. Based on these results, the concentrations for the repetitions were determined.

In the experiment (repetition I and II), the highest nominal applied concentration (62.5 µg/mL) was chosen based on the results obtained in the CRFT. A geometric series (factor 1.2) of eleven dilutions thereof was prepared. Precipitation of the test item was not visible in any of the repetitions.

DMSO (final concentration: 1 %) was used as solvent control and medium no. 3 as growth control. Lactic acid (5000 µM) was used as negative control and EGDMA (120 µM) as positive control.

No substantial and reproducible dose-dependent increase in luciferase induction equal or above 1.5 fold was observed in both repetitions up to the maximal tested non-cytotoxic concentration of the test item. All the validity criteria were fullfilled.

Therefore, the test item was negative in the LuSens assay and is considered not to have the potential to activate the Nrf2 transcription factor under the experimental conditions of this study.