Reaction products of diazotised Metanilic Acid coupled with 8-Hydroxyquinoline, subsequently metalized with Chromium (III) Basic Sulfate and Sodium Formate, 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 09. May. 2018 to 18. May. 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

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:

Draft v. 21. Dec. 2017

Deviations:

yes

Remarks:

see "principles of method if other than guideline"

Principles of method if other than guideline:

Deviations from the Guideline
1. Due to the low solubility of the test item, the preparation differed from the OECD 442D because the additional 1:25 dilution step of the stock solution and the dilutions in medium no. 3 was skipped.
In the CRFT first, a 4 * top dose stock solution in medium no. 3 was prepared and sonicated for 30 min. Afterwards the solution was sterilised by filtration through a low binding nylon filter (pore size 0.2 µm).
2. In both experiments none of the tested concentrations induced a cytotoxic effect.
3. The test item was not considered as inconclusive.
4. The experiments were not repeated.
The deviations 2., 3. and 4. were considered as valid uncritical due to the following reasons:
a) The viability values in the highest test item concentrations were only slightly above the threshold of 70 % (experiment I: 71.8 %, experiment II: 73.7 %)
b) In both experiments a negative trend concerning the luciferase induction was observed at the higher test item concentrations.
c) A definition of the highest test item concentration for the experiments is hardly possible according to the results of the CRFT since the viability values between the concentrations 125 1.0 µg/mL and 2000 125 µg/mL in the pre-test varies only around 18 % (71.7 % - 88.9 %). In addition no dose-dependent effect was detected. Even at the test item concentration 250 µg/mL the viability was still 62.5 %. Just at the higher test item concentrations the viability values falls strongly below 70 %. But since all viability values between the concentrations 31.25 µg/mL and 125 µg/mL are only very slightly above 70 % and small variations of the viability values between individual tests are completely normal (biological system, small changes in experimental performance like number of seeded cells etc.), the probability that all test item concentrations are would be cytotoxic in an additional experiment with 500 µg/mL as highest test item concentration is very high.
d) The highest test item concentration is considerably higher than the maximal concentration which is prescribed by the guideline (CV75 * 1.2). In this study the CV75 * 1.2-concentration corresponds to 24 µg/mL. A repetition of the pre-test for a more precise definition of the CV75 is not useful in this study since no concentration-dependent effect was measured in the CRFT in this study.
5. In experiment I and II a 32 x top dose stock solution of the test item was prepared and 1:8 diluted in medium no. 3. This solution corresponds to the 4 x stock solution. Therefore the preparation of the test item was not performed according to OECD. The deviation was considered as uncritical since the final concentrations in the plate during treatment are not affected by this deviation. Therefore, the deviation has no influence on the final result of the study.

All the deviations were assessed and signed by the deputy study director.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

Acid Orange 061 (a)

IUPAC Name:

Acid Orange 061 (a)

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 (DMEM). Even after sonication, the test item is insoluble in both solvents at the required concentration of 200 mg/mL. At a concentration of 100 mg/mL the test item is soluble in medium.
Due to the low solubility of the test item, the preparation differed from the OECD 442D because the additional 1:25 dilution step of the stock solution and the dilutions in medium no. 3 was skipped.
In the CRFT first, a 4 * top dose stock solution in medium no. 3 was prepared and sonicated for 30 min. Afterwards the solution was sterilised by filtration through a low binding nylon filter (pore size 0.2 µm). In the experiments, a 32 * top dose solution in medium no. 3 was prepared and sonicated for 30 min. Afterwards the solution was sterilised by filtration through a low binding nylon filter (pore size 0.2 µm) and 1:8 prediluted (4 * top dose stock solution).
These stock solutions were used to prepare the geometric series of solutions (CRFT: factor 2, experiments: 1.2). Afterwards 50 µL of each solution was added to 150 µL medium no. 3 containing the cells. 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 (Ludwigshafen, 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, Aa-chen (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 characteris-tics of the cells.
For the Cytotoxicity Range Finder Assay cells of passage 8 were used. For both main experiments cells of passage 10 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 LuSens test at LAUS GmbH was demonstrated in a proficiency study. In this study, 22 proficiency chemicals (indicated by the OECD 442D guideline as well as the OECD PERFORMANCE STANDARDS FOR ASSESSMENT OF PROPOSED SIMILAR OR MODIFIED IN VITRO SKIN SENSITISATION ARE-NRF2 LUCIFERASE TEST METHODS) were tested.

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 main experiments. 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 there-fore 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:
1.0 μg/mL, 2.0 μg/mL, 3.9 μg/mL, 7.8 μg/mL, 15.6 μg/mL, 31.25 μg/mL, 62.5 μg/mL, 125 μg/mL, 250 μg/mL, 500 μg/mL, 1000 μg/mL, 2000 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 until the cells detached. To stop this reaction, medium no. 2 was added. After centrifugation (5 min at 380 * g), the supernatant was discarded 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 humid-ified atmosphere for 24 h and 45 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, 3 wells were used as negative control and 2 wells were used as positive control and 1 well was used as blank control. The plate was sealed with breathable tapes to avoid evaporation 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 incubated 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 (draft version 21. Dec 2017), 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 experiment I and II 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 following 12 nominal concentrations were chosen for experiment I and II:
33.6 μg/mL, 40.4 μg/mL, 48.5 μg/mL, 58.1 μg/mL, 69.8 μg/mL, 83.7 μg/mL, 100.5 μg/mL, 120.6 μg/mL, 144.7 μg/mL, 173.6 μg/mL, 208.3 μg/mL, 250 μg/mL

In the main experiments, a reduction of the viability below 70 % is considered as cytotoxic and is not allowed to be evaluated for luciferase induction.

- Experimental Parameters of Experiment I and II
Experiment I and II were performed in the same way. Experiment II serves only to confirm the results of experiment I.
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 centrifugation (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 experiment I and 25 h 45 min in experiment 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 removed 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 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 transferred to a white flat bottom 96 well plate and the luminescence was measured for 2 seconds 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

Positive control results:

The positive control induced a clear effect with an induction value of 5.2 and 5.6 fold in comparison to the solvent control in experiment I and II respectively.

In vitro / in chemico

Results

Run / experiment:

other: Experiment I and II, test item concentrations (33.6 μg/mL, 40.4 μg/mL, 48.5 μg/mL, 58.1 μg/mL, 69.8 μg/mL, 83.7 μg/mL, 100.5 μg/mL, 120.6 μg/mL, 144.7 μg/mL, 173.6 μg/mL, 208.3 μg/mL, 250 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:

DEMONSTRATION OF PROFICIENCY
As prescribed by the guidelines, more than 80 % (96 %) of the results were correctly categorized. Therefore, the proficiency of the LuSens test was demonstrated.
For all control substances historical data are available, which demonstrates the reliability and the validity of those substances.

- Cytotoxicity Range Finder Test
No cytotoxic effect was observed at the controls as well as the test item concentrations 1.0 μg/mL to 125 μg/mL. The viability values between the concentrations 1.0 μg/mL and 125 μg/mL varies only between 71.7 % and 88.9 %. A dose-dependent effect was not observed. Cytotoxic effects were determined at the test item concentrations 250 μg/mL up to 2000 μg/mL.
The CV75 value was calculated and is 24 μg/mL. It has to be mentioned that the calculation of the CV75 value is not meaningful in this study since no concentration dependent effect was observed at the lower test item concentrations.
The test was valid and could be used for the determination of the concentrations to be used for the main experiments.

- Experiment I
All control substances indicated the expected effect. No considerable reduction of the viability was detected (all values ≥ 87 %). 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: 1.0 fold, negative control: 1.1 fold). However, the positive control induced a clear effect with an induction value of 5.2 fold in comparison to the solvent control.
No cytotoxic effect was observed at any of the test item concentrations. The viability values were all > 71 % and therefore analysable for luciferase induction.
In the Luciferase assay, none of the tested non cytotoxic concentrations induced an increase in 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 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: 1.0 fold, negative control: 1.0 fold). However, the positive control induced a clear effect with an induction value of 5.6 fold in comparison to the solvent control.
No cytotoxic effect was observed at any of the test item concentrations. The viability values were all > 73 % and therefore analysable for luciferase induction.
In the Luciferase assay, none of the tested non cytotoxic concentrations induced an increase in 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.

Experiment I
-Positive control: Fold induction: 5.2; Relative viability: 87.1 %
-Negative control: Fold induction:1.1; Relative viability: 105.2 %
-Growth control: Fold induction: 1.0 ; Relative viability: 101.1 %
-Average percentage standard deviation:18.30 %
- 12 concentrations are analysable
- concentrations cytotoxic (negative result): no
Experiment II
-Positive control: Fold induction: 5.6; Relative viability: 85.3 %
-Negative control: Fold induction: 1.0 ; Relative viability: 103.0 %
-Growth control: Fold induction: 1.0; Relative viability: 99.2 %
-Average percentage standard deviation: 6.48 %
- 12 concentrations are analysable
- concentrations cytotoxic (negative result): no
All validity criteria except the last one were met. But since this deviation concerning the cytotoxic test item concentration was considered as uncritical (see "principles of method if other than guideline") , the study is considered as valid.

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 con-centrations whereby at least three tested concentrations must be non-cytotoxic in two independent valid experiments.
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 molecu-lar weight) and for which no cytotoxicity is observed in any of the tested concentration should be considered as inconclusive.
In order to come to a conclusion on the skin sensitization hazard of a substance, a minimum of two valid and independent experiments needs to indicate a positive or negative result according to the above-described criteria. If the first two experiments come to the same result (i.e. either being negative or being positive) no further testing is required. In case that the first two experiments give discordant results (i.e. one is negative and the other is posi-tive), a third independent experiment 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 experiments are negative/positive, the substance is considered as negative/positive.
The luciferase induction was not above 1.5 fold in more than 2 consecutive non-cytotoxic test item concentrations in experiment I and II.
In both experiments none of the tested concentrations induced a cytotoxic effect. Because of the following reasons this deviation was considered as uncritical and the test was considered as valid: The viability values in the highest test item concentrations were only slightly above the threshold of 70 % and in both experiments a negative trend concerning the lucif-erase induction was observed at the higher test item concentrations. Furthermore a defini-tion of the highest test item concentration for the experiments was hardly possible according to the results of the CRFT. Moreover, the highest test item concentration is considerably higher than the maximal concentration which is prescribed by the guideline (CV75 * 1.2).
Therefore, the test item is considered not to have the potential to activate the Nrf2 transcription factor (no sensitizing potential) under the conditions of this study.

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	2.07	2.07
Growth Control	-	94.2	2.29	2.43
Negative Control	5000 µM	101.5	1.76	1.74
Positive Control	80 µM	72.7	0.88	1.21
Test item	1.0	82.1	0.60	0.73
Test item	2.0	82.3	2.37	2.88
Test item	3.9	88.9	4.79	5.39
Test item	7.8	84.9	6.61	7.79
Test item	15.6	78.6	4.74	6.04
Test item	31.25	71.7	5.07	7.08
Test item	62.5	74.4	2.85	3.83
Test item	125	72.1	1.97	2.73
Test item	250	62.5	2.59	4.14
Test item	500	52.8	1.75	3.32
Test item	1000	35.7	1.28	3.60
Test item	2000	13.9	2.04	14.70

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	0.18	18.30	100.0	2.19	2.19
Growth Control	-	1.0	0.19	19.40	101.1	2.79	2.76
Negative Control	5000 µM	1.1	0.06	5.69	105.2	1.45	1.38
Positive Control	80 µM	5.2	0.52	9.96	87.1	3.53	4.05
Test item	33.6	0.8	0.11	14.31	81.1	2.31	2.85
Test item	40.4	0.8	0.10	12.05	81.3	4.74	5.83
Test item	48.5	0.9	0.07	7.53	81.0	5.77	7.13
Test item	58.1	1.0	0.10	10.07	81.2	5.74	7.06
Test item	69.8	1.1	0.02	2.23	80.4	4.37	5.43
Test item	83.7	1.2	0.10	8.48	81.8	4.71	5.76
Test item	100.5	1.1	0.14	12.18	78.9	4.96	6.29
Test item	120.6	1.2	0.05	4.36	76.4	1.45	1.90
Test item	144.7	1.1	0.08	7.38	75.2	2.21	2.94
Test item	173.6	1.2	0.01	1.07	71.3	1.72	2.41
Test item	208.3	0.7	0.15	21.83	71.6	3.60	5.02
Test item	250.0	0.4	0.07	15.58	71.8	1.01	1.41

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	0.06	6.48	100.0	4.74	4.74
Growth Control	-	1.0	0.04	4.04	99.2	4.97	5.01
Negative Control	5000 µM	1.0	0.06	6.19	103.0	2.20	2.13
Positive Control	80 µM	5.6	0.19	3.40	85.3	2.77	3.25
Test item	33.6	0.9	0.06	7.13	86.9	1.07	1.23
Test item	40.4	0.9	0.08	9.55	84.1	2.23	2.65
Test item	48.5	0.9	0.01	1.30	85.2	1.80	2.11
Test item	58.1	1.0	0.03	3.54	86.3	2.02	2.34
Test item	69.8	1.2	0.04	3.41	86.1	0.68	0.79
Test item	83.7	1.3	0.06	4.52	88.2	8.94	10.14
Test item	100.5	1.4	0.03	2.49	82.4	0.61	0.75
Test item	120.6	1.4	0.06	4.40	86.1	6.77	7.86
Test item	144.7	1.3	0.11	8.11	79.3	1.82	2.30
Test item	173.6	1.3	0.03	2.34	82.0	1.78	2.17
Test item	208.3	1.3	0.10	7.80	74.8	2.55	3.41
Test item	250.0	1.2	0.04	3.13	73.7	3.36	4.55

Historical Data of the Negative Control and the Positive Control

	Σ	Mean induction value	Standard	Range	Present Study
			deviation
					Experiment I	Experiment II
Positive Control	16	6.5	1.408	4.0 - 9.9	5.2	5.6
(p-Phenylene-diamine)
Negative control	162	1.0	0.081	0.8 - 1.3	1.1	1.0
(DL-Lactic acid)

Nominal and Real Test Item Concentrations

Cytotoxicity Range Finder Tests (CRFT)		Experiment I		Experiment 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]
1.0	1.0	33.6	33.6	33.6	33.6
2.0	2.0	40.4	40.3	40.4	40.4
3.9	3.9	48.5	48.4	48.5	48.4
7.8	7.8	58.1	58.1	58.1	58.1
15.6	15.6	69.8	69.7	69.8	69.7
31.3	31.3	83.7	83.6	83.7	83.7
62.5	62.6	100.5	100.3	100.5	100.4
125	125	120.6	120.4	120.6	120.5
250	250	144.7	144.5	144.7	144.6
500	501	173.6	173.4	173.6	173.5
1000	1002	208.3	208.0	208.3	208.2
2000	2003	250.0	249.6	250.0	249.9

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 ( Draft v. 21. Dec. 2017).

The assay included a cytotoxicity range finder test (CRFT) and two independent experiments (experiment 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 the results of this test the concentrations for the two experiments were determined.

In the experiments, the highest nominal applied concentration (250 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 experiments.

Medium No. 3 (final concentration: 1 %) was used as solvent control and as growth control. Lactic acid (5000 μM) was used as negative control and p-Phenylenediamine (80 μM) as positive control.

None of the real treatment concentrations in both experiments deviated more than 10 % from the nominal concentration. Precipitation of the test item was not visible up to the highest concentration.

The viability in the positive control was above 70 % and a distinct increase in luciferase induction above 2.5 fold in comparison to the solvent control was detected. This luciferase induction is well within the historical data range of the positive control.

The viability of negative control was above 70 % and the induction of the luciferase was < 1.5 fold in comparison to the solvent control and well within the historical data range of the negative control.

The induction of the luciferase of the growth control (Medium no. 3) was < 1.5 fold.

In both experiments none of the tested concentrations induced a cytotoxic effect. But since this deviation was considered as uncritical, the study is considered as valid.

No cytotoxic effect was observed in all tested test item concentrations. Therefore, all tested concentrations could be evaluated for luciferase induction.

In all tested concentrations no increase ≥ 1.5 fold in luciferase induction or a concentration-related effect was measured. In addition, in both experiments a negative trend was observed at the higher test item concentrations.

Both experiments are clearly negative. Therefore, the test item was negative in the LuSens assay under the experimental conditions of this study and it is considered not having the potential to activate the Nrf2 transcription factor.