Reaction products of 6-hydroxy-5-nitrosonaphthalene-2-sulfonic acid, chelated with iron (3+), 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 May 27 to June 30, 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:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted February 2015

Deviations:

yes

Remarks:

The used cell line, negative and the positive control, test performance of the BASF protocol is different to the ones of OECD 442D guideline. All deviations are declared as uncritical. The end result is not affected by those changes.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: BASF SE: Protocol LuSens Assay

Version / remarks:

Last update: 16. May 2014 (based on Ramirez et al. 2016)
Ramirez T, Stein N, Aumann A, Remus T, Edwards A, Norman KG, Ryan C, Bader JE, Fehr M, Burleson F, Foertsch L, Wang X, Gerberick F, Beilstein P, Hoffmann S, Mehling A, van Ravenzwaay B, Landsiedel R (2016). Intra- and inter-laboratory re-producibility and accuracy of the LuSens assay: A reporter gene-cell line to detect keratinocyte activation by skin sensitizers. Toxicol In Vitro. 32:278-86.

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD (22. May 2015). PERFORMANCE STANDARDS FOR ASSESSMENT OF PROPOSED SIMILAR OR MODIFIED IN VITRO SKIN SENSITISATION ARE-NRF2 LUCIFERASE TEST METHODS. ENV/JM/MONO(2015)6.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

Acid Green 001

IUPAC Name:

Acid Green 001

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored in the test facility at room temperature (20 ± 5°C) protected from light.
- Solubility and stability of the test substance in the solvent/vehicle: performed to verify whether the test item was sufficiently soluble in DMSO. The solubility test was performed under non-GLP conditions. The test item was soluble at the required concentration of 40 mg/ml.

In vitro test system

Details on the study design:

TEST SYSTEM
- Cell line used: the LuSens cell line was specially designed for this test system by the BASF (Ludwigs-hafen, Germany). It employs the use of a reporter gene for luciferase 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.
- Cell cultures: for mycoplasma contamination screened stocks of LuSens cells are stored in liquid nitrogen in the cell bank of LAUS GmbH to allow a continuous stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells. For the Cytotoxicity Range Finder Assay cells of passage 11 were used. For the experiments cells of passage 13 respectively, were used. After thawing the cells were cultivated in DMEM (9 % FCS) in cell culture flasks at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.
- Test equipment: all vessels used were made of glass or sterilizable plastic. In case of non-sterilization by the manufacturer, they were sterilized before usage in a heating chamber or autoclave. The test was performed in 96-well plates. For the transfer of the culture medium, pipettes were used. Glass measuring flasks and cylinders with conformity sign and standard laboratory material were also used.
- Medium: culture base medium DMEM (Supplier Biochrom AG, 12247 Berlin, Germany) serving as base for Medium no. 2 (500 ml DMEM + 50 ml FCS) and Medium no. 3 (500 ml DMEM + 5 ml FCS).

CYTOTOXICITY RANGE FINDING TEST (CRFT): performed in order to determine the concentration range applicable for experiment I and II. In the CRFT cytotoxicity was determined by measuring the cell viability with MTT. 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: 0.2 µg/ml, 0.4 µg/ml, 0.8 µg/ml, 1.6 µg/ml, 3.1 µg/ml, 6.3 µg/ml, 12.5 µg/ml, 25 µg/ml, 50 µg/ml, 100 µg/ml, 200 µg/ml, 400 µg/ml. The exposure time was 48 h. At the time of seeding the cells were 90 % confluent. The cells were washed twice with PBS (without Ca2+/Mg2+) containing 0.05 % EDTA. Afterwards the cells were trypsinized (by adding Trypsin/EDTA) 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 a clear flat bottom of a 96 well plate. The plate was incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere for 23 h and 40 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). 12 wells were used as solvent control, 6 wells were used as growth control, 3 wells were used as negative control, 2 wells were used as positive control and 1 well was used as blank. 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. All solutions were removed from the wells of the 96 well plate and 200 µl MTT working solution was added to each well. The plates were 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 add-ed to each well. The plate was agitated for 5 min before it was measured at 570 nm and at 690 nm at the photometer.
-Results: no cytotoxic effect was observed at the controls as well as the test item concentrations 0.2 µg/ml to 200 µg/ml. The viability values were all above 81 %. Only at the maximum test item concentration (400 µg/ml) the viability was reduced below 70 % (69.3 %). The test was valid and could be used for the determination of the concentrations to be tested in the main experiments.

CONTROLS: the solutions were freshly prepared on the day of the experiment; tested at only one concentration.
- Negative control: DL-Lactic acid (CAS No: 50-21-5) in DMSO; final concentration: 5000 μM.
- Positive control: EGDMA (Ethylene glycol dimethylacrylate) (CAS No: 97-90-5); final concentration 120 μM.
- Solvent control: DMSO (CAS no: 67-68-5); final concentration: 1 %.
- Growth control: medium no. 3

TEST CHEMICAL CONCENTRATIONS
- Stock solution: on the 1st day of the experiment, a stock solution containing 40 mg/ml (nominal) of the test item in DMSO was prepared. Afterwards this stock solution was filtrated through a hydrophilic Nylon Membrane Filter with a 0.2 µm pore size.
- Test chemical concentrations used: 54 µg/ml, 65 µg/ml, 78 µg/ml, 93 µg/ml, 112 µg/ml, 134 µg/ml, 161 µg/ml, 193 µg/ml, 231 µg/ml, 278 µg/ml, 333 µg/ml, 400.0 µg/ml.
- Dose selection for Experiment I and II : the highest nominal applied concentration (400 µg/ml) was chosen based on the results obtained in the CRFT. A reduction of the viability below 70 % is considered as cytotoxic and is not allowed to be evaluated for luciferase induction. Precipitation of the test item was not visible in any of the experiments.
- Geometric factor: 2 (CRFT) and 1.2 (experiment I and II).
- Solvent used: DMSO.

EXPERIMENTAL PARAMETERS OF EXPERIMENT I AND II
- Preparation of cells: 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. 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 all wells except H12 (blank) of two clear flat bottom 96 well plates. Both plates (one for viability and one for luciferace induction measurement) were incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere for 24 h and 30 min in experiment I and 23 h and 45 min in experiment II.
- Viability measurement: after the incubation time all solutions were 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 for test item concentrations). 12 wells were used as growth control. 24 wells were used as solvent control, 6 wells were used as negative control and 5 wells were used as positive control. 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. 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 plates were 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 was meas-ured by the reduction of the tetrazolium dye MTT (3-(4,5-Dimethyl thiazole 2-yl)-2,5-diphenyltetrazolium-bromide) (yellow color) to its insoluble formazan (purple color) in living cells and therefore indicates the amount of living cells.
- Luciferase induction measurement: for the evaluation of the luciferase expression the second plate was used. The medium was 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 given to the cells and incubated for 5 min at room temperature. During this process the plate was slightly moved. The Steady-Glo® Reagent was prepared by mixing Steady-Glo®-Substrate with Steady-Glo®-Buffer. After lysis 100 µl Steady-Glo® Reagent were added to each well and the plate was shaken 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.

CALCULATION OF LUCIFERASE INDUCTION AND RELATIVE VIABILITY
The calculation of the relative Viability [%] was performed as follows:
All measured wells were corrected = OD570 Value - OD690 value
For the following calculation only the corrected values are used. Relative viability (%) = [(value of well of interest - blank)/(mean solvent control - blank)] * 100 %
Fold induction= (RLU value from measurement - RLU value of blank (only medium without cells))/ (Mean RLU value solvent - RLU value of blank (only medium without cells))
(RLU = Relative Light Unit)
Mean fold induction (Imax, rounded to one decimal place) = (Fold induction of replicate 1 + Fold induction of replicate 2 + Fold induction of replicate 3)/3

EVALUATION CRITERIA
Each valid experiment (i.e. meeting all acceptance criteria) Is interpreted as follows:
- In accordance with the BASF protocol:
A test compound is considered to have the potential to activate the Nrf2 transcription factor if the luciferase induction is above or equal to 1.5 fold compared to the vehicle control in 2 (or more than) consecutive non-cytotoxic tested concentrations whereby at least three tested concentrations must be non-cytotoxic. A test compound is considered not to have the potential to activate the Nrf2 transcription factor if the above effects are not observed.
- In accordance with OECD 442D guideline:
A test compound is considered to have the potential to activate the Nrf2 transcription factor if the luciferase induction is above 1.5 fold compared to the vehicle control and statistically significantly different as compared to the solvent control in a non-cytotoxic tested concen-tration < 1000 μM (or < 200 μg/ml for test chemicals with no defined molecular weight). In addition there must be an apparent overall dose-response for luciferase induction (or a biphasic response in two independent experiments). In addition, a negative result obtained with concentrations < 1000 μM (or < 200 μg/ml for test chemicals with no defined molecular weight) is considered as inconclusive.
In order to come to a conclusion on the potential to activate the Nrf2 transcription factor 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 positive), a third independent experiment needs to be conducted to complete the experiment. The skin sensitizing potential 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.

Results and discussion

Positive control results:

Experiment 1: the positive control induced a clear effect with an induction value of 5.2 fold in comparison to the solvent control
Experiment 2: the positive control induced a clear effect with an induction value of 7.0 fold in comparison to the solvent control.

In vitro / in chemico

Results

Results 1

Run / experiment:

other: Experiment I

Parameter:

other: Luciferase Induction fold

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The viability values were all ≥ 71 %. Therefore all concentrations were analysable for luciferase induction.

Remarks:

None of the tested concentrations induced a luciferase induction above the threshold of 1.5 fold in comparison to the solvent control.

Results 2

Run / experiment:

other: Experiment II

Parameter:

other: Luciferase Induction fold

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: A cytotoxic effect was measured at the concentrations 400 µg/ml and 333 µg/ml. Those concentrations are not included in the final evaluation. In none of the other test item concentrations a cytotoxic effect was detected.

Remarks:

None of the non-cytotoxic tested concentrations induced a luciferase induction above the threshold of 1.5 fold in comparison to the solvent control.

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: induction fold: 1.0 (Experiment I and II); historical data range: 0.8-1.3. No considerable reduction of the viability was detected (relative viability: 107.9 %- Exp I, relative viability = 108.1 % - Exp II).
- Acceptance criteria met for positive control: induction fold: 5.2 (Experiment I), 7.0 (Experimet II); historical data range: 3.8 - 14.7.
- Average percentage standard deviation (luciferase induction) of the variability in at least 21 solvent control wells: below 20 % (7.47 %- Exp I, 6.70 % - Exp II).
- At least 3 test concentrations must be within viability limits, i.e. have relative viability of at least 70 %: 12 (Exp I) and 10 (Exp II) concentrations are analysable.

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

Any other information on results incl. tables

Results of experiment I:

		Induction of Luciferace			Viability of the cells
Parameter	Concentration (μg/ml)	Induction fold	Standard deviation	Standard deviation (%)	Relative viability (%)	Standard deviation	Standard deviation (%)
Solvent control	-	1.0	0.07	7.47	100.0	4.21	4.21
Growth control	-	1.0	0.09	8.73	141.9	4.38	3.09
Negative control	5000 μm	1.0	0.06	5.59	107.9	5.70	5.28
Positive control	120 μm	5.2	0.16	3.04	88.0	1.76	2.00
Test item	54	1.1	0.06	5.41	87.8	2.22	2.53
Test item	65	1.0	0.04	3.66	86.2	2.08	2.42
Test item	78	1.0	0.04	3.91	86.8	2.75	3.17
Test item	93	1.0	0.05	4.65	87.5	3.36	3.84
Test item	112	1.0	0.02	1.66	90.9	3.57	3.93
Test item	134	1.0	0.06	6.03	89.7	3.76	4.19
Test item	161	1.0	0.06	6.27	86.5	3.06	3.54
Test item	193	1.1	0.03	2.54	85.3	4.69	5.50
Test item	231	1.1	0.06	5.90	82.3	4.66	5.66
Test item	278	1.1	0.03	2.64	80.3	3.84	4.77
Test item	333	1.1	0.05	4.31	75.2	2.18	2.90
Test item	400	1.1	0.08	7.17	71.1	2.37	3.33

Results of Experiment II

		Induction of Luciferace			Viability of the cells
Parameter	Concentration (μg/ml)	Induction fold	Standard deviation	Standard deviation (%)	Relative viability (%)	Standard deviation	Standard deviation (%)
Solvent control	-	1.0	0.07	6.70	100.0	4.50	4.50
Growth control	-	1.0	0.10	9.93	130.1	7.09	5.45
Negative control	5000 μm	1.0	0.05	4.63	108.1	8.26	7.64
Positive control	120 μm	7.0	0.22	3.13	90.5	7.13	7.88
Test item	54	1.1	0.06	5.86	97.7	2.77	2.83
Test item	65	1.0	0.17	17.80	88.3	10.36	11.73
Test item	78	0.9	0.10	10.61	86.3	8.33	9.65
Test item	93	1.0	0.02	1.66	87.5	11.86	13.56
Test item	112	1.0	0.06	5.85	88.4	7.93	8.97
Test item	134	1.0	0.08	7.23	88.0	9.40	10.68
Test item	161	1.1	0.07	6.68	84.1	3.74	4.45
Test item	193	1.1	0.02	2.25	79.8	1.49	1.87
Test item	231	1.1	0.02	2.04	78.8	3.09	3.92
Test item	278	1.2	0.10	8.15	73.0	3.31	4.54
Test item	333	1.2*	0.12	9.63	69.5	0.30	0.43
Test item	400	1.1*	0.07	6.25	62.1	0.92	1.49

* = Due to cytotoxicity values will not be used for final evaluation

Applicant's summary and conclusion

Interpretation of results:

other: not classified for skin sensitisation according to the CLP Regulation (EC) No.1272/2008

Conclusions:

The substance does not present a skin sensitisation potential under the conditionds of the LuSens Test.

Executive summary:

The potential of the substance to activate the Nrf2 transcription factor, by using the LuSens cell line was evaluated according to the OECD guideline 442D and the BASF SE: Protocol LuSens Assay. For this purpose, one valid cytotoxicity range finder test (CRFT) and two independent valid experiments (experiment I and II) with a treatment period of 48 h were performed. The CRFT was performed to detect a potential cytotoxic effect of the test item; based on the results 12 test concentrations for the two main experiments were determined to be as:: 54 μg/ml, 65 μg/ml, 78 μg/ml, 93 μg/ml, 112 μg/ml, 134 μg/ml, 161 μg/ml, 193 μg/ml, 231 μg/ml, 278 μg/ml, 333 μg/ml, 400.0 μg/ml. None of the measured treatment concentrations in both experiments deviated more than 10 % from the nominal concentrations. Precipitation of the test item was not visible up to the highest concentration. EGDMA (120 μM) was used as positive control, DL-lactic acid (5000 μM) was used as negative control. Medium No.3 was used as a growth control.

In experiment I no significant reduction of growth was observed in all tested test item concentrations. Therefore, all tested concentrations could be evaluated for luciferase induction. In experiment II a cytotoxic effect was observed at 400 μg/ml and 333 μg/ml. Those concentrations were excluded from the final evaluation of the luciferase induction.

In all tested concentrations of the test item no substantial and reproducible dose dependent increase of luciferase induction was measured. 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 conclusion, under the experimental conditions reported, the test item did not induce the luciferase activity in the LuSens cells above the threshold and the substance does not have the potential to activate the Nrf2 transcription factor according to the OECD Guideline 442D and the BASF protocol criteria.