Alkali metal tetrakis (perfluoro-alcoholato)metallate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan. - Feb. 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Test material form:

solid

Details on test material:

Test material was sublimed for purification.

Specific details on test material used for the study:

The test item was stored in the test facility in a closed vessel at room temperature (20 ±5 °C), kept under inert gas.

In vitro test system

Details on the study design:

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 soluble in DMSO at the required concentration (200 mM). Therefore, 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 3 mM (experiment) test item in DMSO was prepared. Subsequent dilution to 1 % finally yielded a maximum concentration of 2000 µM in the CRFT and 3 µM in the experiment.
For that, the stock solution was first used to prepare a geometric series of solutions (CRFT: factor 2; main experiment: 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 in DMSO as solvent (final concentration: 5000 µM); the solution was freshly prepared on the day of treatment.
Positive Control: EGDMA (Ethylene glycol dimethylacrylate), CAS no. 97-90-5 in DMSO as solvent (final concentration: 120 µM); the solution was freshly prepared on the day of treatment.
Solvent Control: DMSO, CAS no. 67-68-5, purity: 99.5 % (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. 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, Aachen (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 laboratories’ cell bank to allow a continuous stock of cells (mycoplasma contamination free), which guarantees similar parameters of the experiment and reproducible characteristics of the cells. For the Cytotoxicity Range Finder Assay cells of passage 4 were used. For the repetitions, cells of passage 6 and 8 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.
Test vessels: 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.
Chemicals and Media: The purity of the chemicals which were used was either “analytical grade” or “for microbiological purposes”.
Ca2+/Mg2+ solution for PBS: CaCl2 * 2H2O 662.3 mg, MgCl2 * 6H2O 500 mg and H2O demin. ad to 10 mL
EDTA Solution (250 g/L): EDTA dipotassium salt dihydrate 3.46 g, H2O demin. Add to 10 mL
FCS (Fetal Calf Serum) Superior: Ready to use, Supplier Biochrom AG, 12247 Berlin, Germany
Lysepuffer Glo Lysis Buffer 1X: Ready to use, Supplier Promega, Mannheim, Germany.
Medium: Culture base medium DMEM, Supplier PAN-Biotech, 94501 Aidenbach, Germany serving as base for: Medium no. 2 (DMEM 500 mL, FCS 50 mL) and Medium no. 3 (DMEM 500 mL, FCS 5 mL).
MTT-Lysis Buffer: SDS 20 g, DMSO 199.2 mL and Acetic acid 100 %0.8 mL
MTT (3-(4,5-Dimethyl thiazole 2-yl)-2,5-diphenyltetrazolium-bromide) solution (5 mg/mL): MTT reagent 100 mg and PBS 20 mL
MTT-Working solution: MTT Solution 2 mL and Medium No. 3 18 mL
PBS: Phosphate buffered saline, ready to use, Supplier PAN-Biotech, 94501 Aidenbach, Germany
PBS + EDTA solution: PBS 500 mL and EDTA solution (250 g/L) 1 mL
PBS + Ca2+ / Mg2+: PBS 500 mL and Ca2+ / Mg2+ solution 1 mL
Steady-Glo® Reagent: Steady-Glo® Substrate (lyophilized), batch 0000363230 full volume of 1 vial (≙ 1 mL) and Steady-Glo® buffer, batch 0000363022 full volume of 1 vial (≙ 10 mL)
Trypsin/EDTA: Ready to use, Supplier PAN-Biotech, 94501 Aidenbach, Germany
CASYton: Isotonic solution used for the dilution of cell suspensions for cell counting, ready to use, Supplier OLS GmbH & Co KG, Bremen, Germany
Demonstration of proficiency
Prior to routine use, the validity of the LuSens test at the testing facility was demonstrated in a proficiency study. 22 proficiency chemicals indicated by the OECD 442D (version: 25. June 2018) and the OECD PERFORMANCE STANDARDS FOR ASSESSMENT OF PROPOSED SIMILAR OR MODIFIED IN VITRO SKIN SENSITISATION ARE-NRF2 LU-CIFERASE TEST METHODS (version: 22. May 2015) were tested. The 10 proficiency chemicals which are indicated by the current version of the OECD 442D (version: 25. June 2018) were all included in the proficiency study and were categorized correctly regarding the expected LuSens prediction. The reference range (EC1.5, CV75) was correctly obtained for 8 substances. From 22 proficiency chemicals more than 80 % (95 %) of the results were correctly categorized. Therefore, the proficiency of the LuSens test was demonstrated. For all control substances historical data are available which demonstrate the reliability and the validity of these substances.

Results and discussion

In vitro / in chemico

Results

Results 1

Run / experiment:

other: repetition I

Parameter:

other: conc. [µM] with increase ≥ 1.5 fold in luciferase induction

Value:

2.08 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Run / experiment:

other: repetiton II

Parameter:

other: conc. [µM] with increase ≥ 1.5 fold in luciferase induction

Value:

2.5 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

In repetition I a clear does-response curve of the fold induction was observed up to the test item concentration 2.50 µM. Only at the highest test item concentration (3 µM) the fold induction is slightly reduced in comparison to the next lower one. Nevertheless the induction value at the concentration 3 µM is still above the threshold of 1.5 fold and statistically significant in comparison to the solvent control. Since the difference between the induction folds of concentration 2.5 µM and 3 µM is marginal (0.1) and the viability value of the highest test item concentration is only slightly above the cytotoxic range (71.4 %), this effect is considered as not biologically relevant. In repetition II a dose-response curve of the fold induction was observed. Finally both repetitions are considered as clearly positive.

Any other information on results incl. tables

Results of Repetition I: All control substances indicated the expected effect. No considerable reduction of the viability was detected (all values ≥ 90%). 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: 1.0 fold). However, the positive control induced a clear effect with an induction value of 6.5 fold in comparison to the solvent control.

No cytotoxic effect was observed at any of the test item concentrations. Even at the highest test item concentration the viability was only reduced to 71.4%. Therefore, all tested concentrations are analysable for luciferase induction. In the Luciferase assay, the following test item concentration induced a statistically significant increase in luciferase induction above the threshold of 1.5 fold in comparison to the solvent control: 3.00 µM, 2.50 µM, 2.08 µM

Results of repetition I

		Induction of Luciferase			Viability of the Cells
Parameter	Concentration	Induction	Standard Deviation	Standard Deviation	Relative Viability	Standard Deviation	Standard Deviation
	[µM]	fold		[%]	[%]		[%]
Solvent Control	-	1.0	0.08	7.65	100.0	4.39	4.39
Growth Control	-	1.1	0.11	9.45	133.8	4.96	3.71
Negative Control	5000	1.0	0.07	6.65	111.2	5.01	4.51
Positive Control	120	6.5	0.38	5.85	90.4	2.06	2.28
Test item	0.40	0.9	0.04	3.86	88.4	11.07	12.52
Test item	0.48	0.9	0.05	5.60	87.7	5.72	6.52
Test item	0.58	0.9	0.03	3.46	88.5	1.49	1.68
Test item	0.70	1.0	0.09	9.16	88.4	6.23	7.05
Test item	0.84	1.0	0.14	13.27	88.8	4.24	4.77
Test item	1.00	1.0	0.04	4.19	84.2	6.98	8.29
Test item	1.21	1.1	0.03	2.50	80.0	4.60	5.75
Test item	1.45	1.2	0.11	9.05	80.8	4.45	5.51
Test item	1.74	1.4	0.11	7.71	88.6	1.69	1.91
Test item	2.08	1.8	0.32	17.49	86.3	4.00	4.63
Test item	2.50	3.2	0.61	19.06	88.6	3.47	3.92
Test item	3.00	3.1	0.33	10.49	71.4	1.85	2.59

 

Results of Repetition 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 (growth control: 1.0 fold, negative control: 1.1 fold). However, the positive control induced a clear effect with an induction value of 7.1 fold in comparison to the solvent control.

No cytotoxic effect was observed at any of the test item concentrations. Therefore, all tested concentrations are analysable for luciferase induction. In the Luciferase assay, the following test item concentration induced a statistically significant increase in luciferase induction above or equal the threshold of 1.5 fold in comparison to the solvent control: 3.00 µM, 2.50 µM

Results of repetition II

		Induction of Luciferase			Viability of the Cells
Parameter	Concentration	Induction	Standard Deviation	Standard Deviation	Relative Viability	Standard Deviation	Standard Deviation
	[µM]	fold		[%]	[%]		[%]
Solvent Control	-	1.0	0.05	4.79	100.0	6.41	6.41
Growth Control	-	1.0	0.05	5.48	124.8	6.01	4.82
Negative Control	5000	1.1	0.05	4.67	108.7	5.95	5.47
Positive Control	120	7.1	0.50	6.97	101.7	3.95	3.89
Test item	0.40	0.9	0.02	2.27	88.9	0.61	0.69
Test item	0.48	1.0	0.06	6.46	92.1	4.30	4.67
Test item	0.58	0.9	0.04	4.63	93.5	2.17	2.32
Test item	0.70	1.0	0.02	1.98	91.1	3.15	3.46
Test item	0.84	1.0	0.03	2.95	90.3	1.84	2.04
Test item	1.00	1.0	0.02	2.16	90.4	6.57	7.27
Test item	1.21	1.0	0.07	6.99	91.6	2.53	2.76
Test item	1.45	1.0	0.06	6.01	87.5	2.02	2.31
Test item	1.74	1.1	0.03	2.27	86.7	4.16	4.80
Test item	2.08	1.2	0.08	6.19	88.3	1.95	2.21
Test item	2.50	1.5	0.13	8.87	92.6	4.12	4.45
Test item	3.00	1.7	0.15	8.80	98.8	1.49	1.51

 

Acceptability: In the following table the criteria for acceptability as well as the corresponding results in repetition I and II are given.

Acceptability of repetition I and II

Criteria	Found in repetition I	Found in repetition II
The average induction for the positive control should be ≥ 2.5 fold and it should have a relative viability of at least 70 %.	Positive control Fold induction: 6.5 Relative viability: 90.4 %	Positive control Fold induction: 7.1 Relative viability: 101.7 %
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 %.	Negative control: Fold induction: 1.0 Relative viability: 111.2 % Growth control: Fold induction: 1.1 Relative viability: 133.8 %	Negative control: Fold induction: 1.1 Relative viability: 108.7 % Growth control: Fold induction: 1.0 Relative viability: 124.8 %
The average percentage standard deviation (luciferase induction) of the variability in at least 21 solvent control wells should be below 20 %.	7.65 %	4.79 %
At least 3 test concentrations must be within viability limits, i.e. have relative viability of at least 70 %.	12 concentrations are analysable	12 concentrations are analysable
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	the maximum concentration of 2000 µM (2000 µg/mL) was tested	the maximum concentration of 2000 µM (2000 µg/mL) was tested

 

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

Prediction Model: 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 factorif 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 concentrations whereby at least three tested concentrations must be non-cytotoxic in two independent valid repetitions.

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 molecular 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, a minimum of two valid and independent repetitions need to indicate a positive or negative result according to the above-described criteria. If the first two repetitions come to the same result (i.e. either being negative or being positive) no further testing is required. In case that the first two repetitions give discordant results (i.e. one is negative and the other is positive), a third independent repetition needs to be conducted to complete the study. 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 repetitions are negative/positive, the substance is considered as negative/positive.

The luciferase induction was≥ 1.5 fold and statistically significant compared to the solvent control in2 (or more)consecutive non-cytotoxic tested concentrations in repetition I and II. Therefore, the test item AM(pfa)4 is considered to have the potential to activate the Nrf2 transcription factor under the conditions of the LuSens test.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In conclusion of this OECD 442D study, it can be stated that under the experimental conditions of this study, the test item, AM(pfa)4, was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor.

Executive summary:

This in vitro study evaluates the potential of the test item AM(pfa)4 to activate the Nrf2 transcription factor by using the LuSens cell line and is part of a tiered strategy for the evaluation of skin sensitization potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers sensitisers and non-sensitizers sensitisers in the context of an integrated approach to testing and assessment. The LuSens test is an ARE Reporter Gene Assay performed according to the OECD 442D Guideline with the title “In Vitro Skin Sensitisation assays addressing the AOP Key Event on Keratinocyte activation”.

The assay included a cytotoxicity range finder test (CRFT) and one experiment, consisting of two independent repetitions (repetition 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 these results, the concentrations to be tested in the repetitions were determined.

In the experiment (repetition I and II), the highest nominal applied concentration (3 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 repetitions. DMSO (final concentration: 1 %) was used as solvent control and medium no. 3 as growth control. Lactic acid (5000 µM) was used as negative control and EGDMA (120 µM) as positive control.

In summary a statistically significant and reproducible dose-dependent increase in luciferase induction ≥ 1.5 fold in ≥ two non-cytotoxic consecutive test item concentrations was observed in both repetitions.

Therefore, under the experimental conditions of this study, the test item, AM(pfa)4, was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor.