Acid Brown 354

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from 31. Jan. 2017 to 17. Feb. 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:

2015

Deviations:

yes

Remarks:

see "Any other information of materials and methods"

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: BASF SE: Protocol LuSens Assay

Version / remarks:

Last update: 16. May 2014

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of diazotized 4'-Amino-4-Nitrodiphenylamine-2-Sulfonic Acid coupled with Resorcinol, sodium salts

EC Number:

947-125-5

Molecular formula:

Not applicable for UVCB substance

IUPAC Name:

Reaction products of diazotized 4'-Amino-4-Nitrodiphenylamine-2-Sulfonic Acid coupled with Resorcinol, sodium salts

Test material form:

solid: particulate/powder

In vitro test system

Details on the study design:

PREPARATION OF TEST ITEM
-Solubility test
This test was performed to verify whether the test item is sufficiently soluble in DMSO. The solubility test was performed under non-GLP conditions before starting the experimental phase. The test item was soluble in DMSO at the concentration 40 mg/mL.
-Preparation
On the 1st day of the experiment, a stock solution containing 40 mg/mL (CRFT) and 25 mg/mL (experiment I and II) of the test item in DMSO was prepared. In experiment I and II this stock solution was afterwards diluted (1:10 fold) in DMSO to prepare a stock solution, which was used for the further preparation. The stock solutions were afterwards used to prepare the geometric series (factor 2 (CRFT) and factor 1.2 3 (experiment I and II)) of the resulting test item concentrations. Skin sensitisation (In vitro test system)
- Details on study design:On the 1st day of the experiment, a stock solution containing 40 mg/ml (CRFT) and 25 mg/mL (experiment I and II) of the test item in DMSO was prepared. In experiment I and II this stock solution was afterwards diluted (1:10 fold) in DMSO to prepare a stock solution, which was used for the further preparation. The stock solutions were afterwards used to prepare the geometric series (factor 2 (CRFT) and factor 1.2 3 (experiment I and II)) of the resulting test item concentrations.

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 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 (laboratory of PD Dr. Wruck).
-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 12 were used. For the experiments cells of passage 14 (experiment I) and 6 (experiment II) 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 .

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 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.
- Performance
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 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.
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 was 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 added to each well. The plate was agitated for 5 min before it was measured at 570 nm and at 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 to the OECD guideline 442D and the protocol of the BASF SE, 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 400 µg/mL can also be used. Alternative concentrations may be used upon justification (e.g. in case of cytotoxicity or poor solubility). As solvent, DMSO will be used.
In case of cytotoxic results the highest test item concentration in experiment I and II corresponds to the first concentration indicating viability below 70 % in the CRFT.
Since a cytotoxic reaction was observed in the CRFT the following 12 nominal concentrations were chosen for experiment I and II: 3.36 µg/mL, 4.04 µg/mL, 4.85 µg/mL, 5.81 µg/ml, 6.98 µg/mL, 8.37 µg/mL, 10.05 µg/mL, 12.06 µg/mL, 14.47 µg/mL, 17.36 µg/mL, 20.83 µg/ml and 25 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 24 h in Experiment II.
The treatment with the controls as well as the test item solutions is identical on both plates (one for viability and one for luciferase induction 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 add-ed 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 con-trol, 5 wells were used as positive control and 1 well was used as blank. The arrangement of the substances on the 96 well plate is demonstrated. The plate was sealed with breathable tape 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 performance of the viability assay one of the 2 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 plates were incubated for 2 h min 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 measured 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. After the measurement of the color change, the values were transferred in a validated spreadsheet for the calculation of the viability.
For the performance of the luciferase induction the second plate was used. 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 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, 5 wells were used as positive control and 1 well was used as blank. The arrangement of the substances on the 96 well plate is demonstrated. The plate was sealed with breathable tape 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 evaluation of the luciferase expression 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 tem-perature. Then, 170 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.

Results and discussion

Positive control results:

(Experiment I) The positive control induced a clear effect with an induction value of 4.2 fold in comparison to the solvent control.
(Experiment II) the positive control induced a clear effect with an induction value of 6.8 fold in comparison to the solvent control.

In vitro / in chemico

Results

Results 1

Run / experiment:

other: Experiment I, test item concentrations (3.36-14.47 µg/ml)

Parameter:

other: luciferase induction

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Run / experiment:

other: Experiment II, test item concentrations (3.36-12.06 µg/ml)

Parameter:

other: luciferase induction

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

-Cytotoxicity Range Finder Test
Regarding the controls no critical reduction of the viability was detected. The lowest value was achieved by the positive control, with a viability of 87 %. Therefore, all controls are valid.
No cytotoxic effects were observed at the test item concentrations 0.2 µg/ml - 12.5 µg/ml. The viability values were all ≥ 80 %. At the next higher test item concentration 25 µg/ml the viability was reduced to 55.1 %. This is declared as a cytotoxic effect. In all higher test item concentrations the viability was reduced to ≤ 55 %.

-Experiment I
All control substances indicated the expected effect. No considerable reduction of the viability was detected (all values above 94 %). 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: 1.2 fold, negative control: 1.0 fold).
In experiment I a cytotoxic effect was observed at the test item concentrations 25 µg/ml, 20.83 µg/ml and 17.36 µg/ml. Therefore, those three concentrations were excluded from the final evaluation. None of the other test item concentrations (14.47 µg/ml - 3.36 µg/ml) induced a cytotoxic effect. The viability values were all ≥ 70 %.
Regarding the test item, none of the tested and non-cytotoxic concentrations induced a luciferase induction above the threshold of 1.5 fold in comparison to the solvent control. All induction values were ≤ 1.4 fold.

-Experiment II
All control substances indicated the expected effect. No considerable reduction of the viability was detected (all values ≥ 94 %). 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 (both: 1.0 fold).
In experiment II a cytotoxic effect was observed at the test item concentrations 25 µg/ml - 14.47 µg/ml. Therefore, those four concentrations were excluded from the final evaluation. None of the other test item concentrations (12.06 µg/ml - 3.36 µg/ml) induced a cytotoxic effect. The viability values were all ≥ 79 %.
Regarding the test item, none of the tested and non-cytotoxic concentrations induced a luciferase induction above the threshold of 1.5 fold in comparison to the solvent control. All induction values were ≤ 1.2 fold

Any other information on results incl. tables

VALIDITY CRITERIA FULFILLED

All validity criteria were met. Therefore, the study is valid.

Experiment I

-Positive control

Fold induction: 4.2

Relative viability: 94.0 %

-Negative control:

Fold induction: 1.0

Relative viability: 102.4 %

-Growth control:  

Fold induction: 1.2

Relative viability: 139.3 %

-Average percentage standard deviation: 12.33 %

-9 concentrations are analysable

Experiment II

-Positive control

Fold induction: 6.8

Relative viability: 94.0 %

-Negative control:

Fold induction: 1.0

Relative viability: 107.4 %

-Growth control:  

Fold induction: 1.0

Relative viability: 131.8 %

-Average percentage standard deviation: 10.27 %

-8 concentrations are analysable

CLASSIFICATION

In accordance to the BASF protocol:

The test item is considered “not to have a sensitizing potential under the conditions of the LuSens test”.

In accordance to OECD 442D guideline:

In accordance to the OECD 442D the result is considered as inconclusive.

Applicant's summary and conclusion

Interpretation of results:

other: no potential to activate keratinocytes

Conclusions:

The test item was considered not to have a sensitizing potential under the conditions of the LuSens test in accordance to the BASF protocol.

Executive summary:

In order to investigate the sensitizing potential of test item, an in vitro test was performed using the LuSens cell line. The LuSens test is an ARE (antioxidant response element) Reporter Gene Assay that was developed by the BASF SE (Ludwigshafen, Germany) and is based on the OECD Guideline No. 442D (KerationSens Assay). The assay differs in some points from the OECD guideline. The assay was performed in 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 (25 µg/ml) was chosen with regard to the cytotoxic reaction in the CRFT. Furthermore, a geometric series (factor 1.2) of eleven dilutions was prepared. Precipitation of the test item was not visible in all experimental parts. DMSO (final concentration: 1 %) was used as solvent control and medium no. 3 as medium control. Furthermore, 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 above 1.5 fold was observed in both experiments up to the maximal concentration of the test item.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item possesses no sensitizing potential in accordance to the BASF protocol.