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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
31 January to 17 February, 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 guidelineopen allclose all
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 material and methods"
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

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of Cobalt, 4-[4-[[4-[[[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxyphenyl]sulfonyl]amino]phenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulfonate 4-[4-[[4-[[[3-[[4,5-dihydro-3-methyl-5-oxo-1-(4-sulfophenyl)-1H-pyrazol-4-yl]azo]-4-hydroxyphenyl]sulfonyl]amino]phenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulfonate sodium complexes and Cobaltate(3-), bis[4-[4-[[4-[[[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxyphenyl]sulfonyl]amino]phenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulfonato(3-)]-, trisodium and Cobaltate(5-), bis[4-[4-[[4-[[[3-[[4,5-dihydro-3-methyl-5-oxo-1-(4-sulfophenyl)-1H-pyrazol-4-yl]azo]-4-hydroxyphenyl]sulfonyl]amino]phenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulfonato(4-)]-, pentasodium
EC Number:
916-898-0
Molecular formula:
C64H46CoN18Na5O22S6 C64H47CoN18Na4O19S5 C64H48CoN18Na3O16S4
IUPAC Name:
Reaction mass of Cobalt, 4-[4-[[4-[[[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxyphenyl]sulfonyl]amino]phenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulfonate 4-[4-[[4-[[[3-[[4,5-dihydro-3-methyl-5-oxo-1-(4-sulfophenyl)-1H-pyrazol-4-yl]azo]-4-hydroxyphenyl]sulfonyl]amino]phenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulfonate sodium complexes and Cobaltate(3-), bis[4-[4-[[4-[[[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxyphenyl]sulfonyl]amino]phenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulfonato(3-)]-, trisodium and Cobaltate(5-), bis[4-[4-[[4-[[[3-[[4,5-dihydro-3-methyl-5-oxo-1-(4-sulfophenyl)-1H-pyrazol-4-yl]azo]-4-hydroxyphenyl]sulfonyl]amino]phenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulfonato(4-)]-, pentasodium
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 (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.
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 25.00 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
-Experimental Performance
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. 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 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 temperature. 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 6.2 fold in comparison to the solvent control.
(Experiment II) 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

Resultsopen allclose all
Run / experiment:
other: Experiment I, test item concentrations (17.36 µg/ml - 3.36 µg/ml)
Parameter:
other: luciferase induction
Value:
1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: Experiment II, test item concentrations (25 µg/ml - 3.36 µg/ml)
Parameter:
other: luciferase induction
Value:
1.1
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 82.1 %. 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 ≥ 77 %. At the next higher test item concentration 25 μg/mL the viability was reduced to 61.2 %. This is declared as a cytotoxic effect. In all higher test item concentrations the viability was reduced to ≤ 59 %.

- Experiment I
All control substances indicated the expected effect. No considerable reduction of the viability was detected (all values ≥ 96 %). 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). However, the positive control induced a clear effect with an induction value of 6.2 fold in comparison to the solvent control.
In experiment I a cytotoxic effect was observed at the test item concentrations 25 μg/mL and 20.83 μg/mL. Therefore those two concentrations were excluded from the final evaluation. None of the other test item concentrations (17.36 μg/mL - 3.36 μg/mL) induced a cytotoxic effect. The viability values were all ≥ 77 %.
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 fold.

- Experiment II
All control substances indicated the expected effect. No considerable reduction of the viability was detected (all values ≥ 100 %). 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). However, the positive control induced a clear effect with an induction value of 7.0 fold in comparison to the solvent control.
In this experiment none of the test item concentrations induced a cytotoxic effect. The viability of the highest test item concentration 25 μg/mL was reduced to 78.6 % but still within the non-cytotoxic range.
Regarding the test item, none of the tested concentrations induced a luciferase induction above the threshold of 1.5 fold in comparison to the solvent control. All values were ≤ 1.1 fold.

Any other information on results incl. tables

VALIDITY CRITERIA FULFILLED:

All validity criteria about experiment I and II were met. Therefore, the study is valid.

Experiment I

-Positive control: Fold induction: 6.2; Relative viability: 96.2 %

-Negative control: Fold induction: 1.0; Relative viability: 107.0 %

-Growth control: Fold induction: 1.2; Relative viability: 142.1 %

-Average percentage standard deviation: 7.78 %

-10 concentrations are analysable

Experiment II

-Positive control; Fold induction: 7; Relative viability: 101.9 %

-Negative control: Fold induction: 1.0; Relative viability: 110.9 %

-Growth control: Fold induction: 1.0; Relative viability: 132.4 %

-Average percentage standard deviation: 6.70 %

-12 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 sensitizing potential under the conditions of the LuSens test in accordance to the BASF protocol
Conclusions:
The test item was considered to have no sensitising potential under the conditions of the LuSens test in accordance to the BASF protocol
Executive summary:

In order to investigate the sensitising potential of test item, an in vitro sensitisation test was performed using the LuSens cell line. The LuSens test is an ARE Reporter Gene Assay that was developed by the BASF SE (Ludwigshafen, Germany) and is based on the OECD 442D Guideline (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. 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 2 (120 µM) as positive control.

No substantial or reproducible dose-dependent increase in luciferase induction above 1.5 fold was observed in either experiment, 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 sensitising potential in accordance to the BASF protocol.