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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:
November 6th to 16th, 2018
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
adopted June 25th, 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Series on Testing and Assessment No. 2013: “PERFORMANCE STANDARDS FOR ASSESSMENT OF PROPOSED SIMILAR OR MODIFIED IN VITRO SKIN SENSITISATION ARE-NRF2 LUCIFERASE TEST METHODS.” ENV/JM/MONO(2015)6.
Version / remarks:
adopted May 22nd, 2015
Qualifier:
according to guideline
Guideline:
other: EU-Method B.60 (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
adopted Feb. 14th, 2017
Deviations:
no
Principles of method if other than guideline:
Additional information was taken from:
- Ramirez T, Mehling A, Kolle SN, Wruck CJ, Teubner W, Eltze T, Aumann A, Urbisch D, van Ravenzwaay B, Landsiedel R (2014). LuSens: a keratinocyte based ARE reporter gene assay for use in integrated testing strategies for skin sensitization hazard identification. Toxicol In Vitro. 28(8): 1482-97
- 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 reproducibility and accuracy of the LuSens assay: A reporter gene-cell line to detect keratinocyte activation by skin sensitizers. Toxicol In Vitro. 32:278-86.
- Bauch C, Kolle SN, Ramirez T, Eltze T, Fabian E, Mehling A, Teubner W, van Ravenzwaay B, Landsiedel R (2012). Putting the parts together: Combining in vitro methods to test for skin sensitizing potentials. Regul Toxicol Pharmacol (under revision).
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethyl 4-oxovalerate
EC Number:
208-728-2
EC Name:
Ethyl 4-oxovalerate
Cas Number:
539-88-8
Molecular formula:
C7H12O3
IUPAC Name:
ethyl 4-oxopentanoate

In vitro test system

Details on the study design:
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 sol-uble in DMSO and medium at the required concentration (200 mM). Since DMSO is the preferred solvent according to OECD 442D, DMSO was used as solvent.
The highest test item concentration in the Cytotoxicity Range Finder Test (CRFT) is 2000 μM. Since the final concentration of the solvent during treatment is limited to 1 %, a stock solution containing 200 mM (CRFT and main experiments) test item in DMSO was prepared. Subsequent dilution to 1 % finally yielded a maximum concentration of 2000 μM in the CRFT and the main experiments. For that, the stock solution was first used to prepare a geometric series of solutions (CRFT: factor 2; main experiments: 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.

CONTROLS
- Negative control: DL-Lactic acid, CAS No 50-21-5, Purity: 90.5 % (this purity 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), Final concentration: 5000 μM
- Positive Control: EGDMA (Ethylene glycol dimethylacrylate), CAS No: 97-90-5, Purity: 98 % (this purity 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 requestedtreatment,120 μM of the pure EGDMA is tested), Final concentration: 120 μM
-Solvent Control: Name DMSO, CAS no. 67-68-5, Final concentration: 1 % (in medium no. 3)

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 (Ludwigs-hafen, 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, Aa-chen, 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 myco-plasma contamination screened stocks of LuSens cells are stored in liquid nitrogen in the cell bank 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 10 were used. For both main experiments cells of passage 12 were used. After thawing (> 15 days prior to the treatment), 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.

CHEMICALS AND MEDIA
- Medium no. 2: DMEM 500 ml + FCS 50 ml
- Medium no. 3: DMEM 500 ml + FCS 5 ml
- MTT-Lysis Buffer: SDS 20 g + DMSO 199.2 ml + Acetic acid 100 % 0.8 ml
- MTT-Working Solution: MTT Solution 2 ml + Medium No. 3 18 ml

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 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: 0.98 μM, 1.95 μM, 3.91 μM, 7.81 μM, 15.63 μM, 31.25 μM, 62.5 μM, 125 μM, 250 μM, 500 μM, 1000 μM, 2000 μM. 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 (by add-ing Trypsin/EDTA to the flask) 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 sus-pension 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 23 h 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 (cells+medium no.3), 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 (reference) at the photometer.
- 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 experiment I and II should be determined so that at least one of them is in the cytotoxic range. If no cytotoxic effect is measured in the CRFT the highest test item concentration in experiment I and II should be 2000 μM. The dilution factor in the main experiments is always 1.2 fold. Since no cytotoxic reaction was observed in the CRFT the following 12 nominal concentra-tions were chosen for experiment I and II: 269 μM, 323 μM, 388 μM, 465 μM, 558 μM, 670 μM, 804 μM, 965 μM, 1157 μM, 1389 μM, 1667 μM, 2000 μM. In the main experiments, a reduction of the viability below 70 % is considered as cytotoxic and the concentration that is cytotoxic 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.
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, 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 hu-midified atmosphere for 24 h in experiment I and 23 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 liv-ing cells and therefore indicates the amount of living cells. After the measurement of the colour change, the values were used 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-hr 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 transferred to a white flat bottom 96 well plate and the luminescence was measured for 2 seconds using a luminometer.

Results and discussion

In vitro / in chemico

Results
Run / experiment:
other: other: other: other: Experiment 1 and Experiment II
Parameter:
other: Luciferace fold induction
Value:
1.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Luciferace fold induction
Other effects / acceptance of results:
Experiment I: all control substances indicated the expected effect. No considerable reduction of the viability was detected (all values ≥ 80 %). 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: 1.1 fold, negative control: 0.9 fold). However, the positive control induced a clear effect with an induction value of 4.6 fold in comparison to the solvent control. No cytotoxic effect was observed at all test item concentrations. The viability values were all > 89 % and therefore analysable for luciferase induction. In the Luciferase assay, none of the tested 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 viability was detected (all values ≥ 81 %). 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: 1.1 fold, negative control: 0.9 fold). However, the positive control induced a clear effect with an induction value of 5.0 fold in comparison to the solvent control. No cytotoxic effect was observed at all test item concentrations. The viability values were all > 98 % and therefore analysable for luciferase induction. In the Luciferase assay, none of the tested concentrations induced a luciferase induction above or equal 1.5 fold in comparison to the solvent control.

DEMONSTRATION OF TECHNICAL PROFICIENCY: prior to routine use, the validity of the LuSens test was demonstrated in a proficiency study. 22 proficiency chemicals indicated by the OECD 442 D (status: 25. June 2018) and the OECD PERFORMANCE STANDARDS FOR ASSESSMENT OF PRO-POSED SIMILAR OR MODIFIED IN VITRO SKIN SENSITISATION ARE-NRF2 LUCIFER-ASE TEST METHODS (adoption: 22 May 2015) were tested. The 10 proficiency chemicals which are indicated by the current version of the OECD 442D (status: 25. June 2018) were all included in the proficiency study. From 22 proficiency chemicals more than 80 % of the results were correctly categorized. From the 10 of the proficiency chemicals of the current OECD 442D (chemicals of the current version of the OECD 442 D (status: 25. June 2018) 80 % of the results were correctly categorized. Therefore, the proficiency of the LuSens test was demonstrated.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: yes

Applicant's summary and conclusion

Interpretation of results:
other: The substance is considered not to have the potential to activate the Nrf2 transcription factor
Conclusions:
The substance is considered not to have the potential to activate the Nrf2 transcription factor under the test conditions.
Executive summary:

The potential of the test item to activate the Nrf2 transcription factor (sensitizing potential) was evaluated by using the LuSens cell line according to the OECD Guideline 442D and EU Method B60.

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 (2000 μM) 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. DMSO (final concentration: 1 %) was used as solvent control and medium no. 3 as growth control (blank medium 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 experiments up to the maximal tested concentration of the test item.

Under the experimental conditions of this study, the test item, was negative and is therefore considered not to have the potential to activate the Nrf2 transcription factor.

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