[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From November 22th, 2022 to February 14th, 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

Version / remarks:

30 Jun 2022

GLP compliance:

yes

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Test material

Test material information

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Water

Constituent 1

Reference substance name:

Zinc bis[(1Z)-3-oxo-1,3-diphenyl-1-propen-1-olate]

Cas Number:

21333-45-9

Molecular formula:

C30H22O4.Zn

IUPAC Name:

Zinc bis[(1Z)-3-oxo-1,3-diphenyl-1-propen-1-olate]

Test material form:

solid

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Before use, the test item was homogenized by grinding with mortar and pestle

In vitro test system

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
The highest test item concentration in the KeratinoSens™ test is 2000 µM in repetition I and 31.25 µM in repetition IV and V. Since the final concentration of the solvent during treatment is limited to 1 %, a stock solution of 200 mM (corresponding to 105.17 mg/mL, purity taken into account) (repetition I) and 3.125 mM (corresponding to 1.6 mg/mL) (repetition IV and V) of the test item in DMSO was prepared and mixed for 5 minutes until it was completely dissolved. Subsequent dilution to 1 % finally yielded a maximum concentration of 2000 µM in repetition I and 31.25 µM in repetition IV and V.
For that, the stock solution was first used to prepare a geometric series of solutions (1:2) on a 100 x DMSO Master Plate. Afterwards all concentrations were further diluted (1:25) in medium no. 3 on a 4 x Master Plate. Another 1:4 dilution was achieved by adding 50 µL of each concentration of the 4 x Master Plate to the corresponding wells of the Assay Plates (Viability Assay Plate and Luciferase Assay Plates) 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.


DOSE SELECTION:
In accordance with the OECD guideline 442D, the maximum final test item concentration should be 2000 µM. Alternative concentrations may be used upon justification (e.g. in case of cytotoxicity or poor solubility).
The following 12 nominal concentrations were tested in repetition I:
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
Due to precipitation and a strong cytotoxic effect of the test item in repetition I the concentrations were adapted and the following test item concentrations were tested in repetition IV and V:
0.02 µM, 0.03 µM, 0.06 µM, 0.12 µM, 0.24 µM, 0.49 µM, 0.98 µM, 1.95 µM, 3.91 µM, 7.81 µM, 15.63 µM, 31.25 µM
The real test item concentrations have been calculated..
A test item concentration inducing a viability below 70 % is considered as cytotoxic and is not allowed to be evaluated for luciferase induction.


APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
All repetitions were performed in the same way. At the time of seeding the cells were between 80-90% 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. 3 was added. After quantification the cell suspension was adjusted to 80000 (± 10 %) cells per mL. 125 µL of the cell suspension (approximately 10000 cells) were seeded in all wells except well H12 (Blank) of one clear flat bottom 96 well plate as well as three white flat bottom 96 well plates. Afterwards, the cells were left in the clean bench for 30 min in order that the cells could attach evenly distributed. All four plates were incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere for 24 h.
The treatment procedure was performed on all 96 well plates identically:
After the incubation time the medium was removed from the cells in all four plates and 150 µL medium no. 3 was added to each well. Afterwards 50 µL of each single test item concentration and the controls of the 4 x master plate were added to the corresponding wells of the four test plates containing the cells and the medium. Six wells were used for the solvent control, five wells for positive control and one 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.


LUCIFERASE ACTIVITY MEASUREMENTS
For the evaluation of the viability, the clear flat bottom 96 well plate was used:
All solutions were removed from the wells of the clear 96 well plate and 200 µL MTT working solution were added to each well. The plate was incubated for 4 h at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere. Afterwards the solution was removed and 50 µL Isopropanol was added to each well. The plate was agitated for 30 min before it was measured at 570 nm 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 evaluation of the Luciferase induction, the three white 96 well plates were used:
For the evaluation of the Luciferase expression all solutions were removed from the wells and the cells were washed with 200 µL PBS. Afterwards 100 µL Medium no. 3 and 100 µL Steady-Glo® Reagent were added to each well and the plates were shaken again slowly for 5 min at room temperature. Afterwards the luminescence was measured using a luminometer.
For calculation of the luciferase induction, a validated Microsoft Excel® spreadsheet file was used.

Vehicle / solvent control:

DMSO

Positive control:

cinnamic aldehyde [442D]

Results and discussion

Positive control results:

For the positive control cinnamic aldehyde, historical data are available which demonstrates the reliability and the validity of this substance

In vitro / in chemico

Results

Results 1

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

IC50 [442D]

Value:

6.3 µM

Remarks on result:

other: The result of this repetition was considered “inconclusive” as no-dose dependent effect was observed

Results 2

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

IC50 [442D]

Remarks on result:

not determinable because of methodological limitations

Remarks:

invalid repetition

Results 3

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

IC50 [442D]

Remarks on result:

not determinable because of methodological limitations

Remarks:

invalid repetition

Results 4

Group:

test chemical

Run / experiment:

other: run/experiment 4

Parameter:

IC50 [442D]

Value:

12.4 µM

Remarks on result:

positive indication of skin sensitisation

Results 5

Group:

test chemical

Run / experiment:

other: run/experiment 4

Parameter:

EC 1.5 [442D]

Value:

0.2 µM

Remarks on result:

positive indication of skin sensitisation

Results 6

Group:

test chemical

Run / experiment:

other: run/experiment 5

Parameter:

IC50 [442D]

Value:

6.4 µM

Remarks on result:

positive indication of skin sensitisation

Results 7

Group:

test chemical

Run / experiment:

other: run/experiment 5

Parameter:

EC 1.5 [442D]

Value:

0.2 µM

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

Results of Controls in Repetition I

	SC	SC	SC	SC	SC	SC	PC 4 µM	PC 8 µM	PC 16 µM	PC 32 µM	PC 64 µM
Viability [%]	91	98	87	98	112	115	85	93	102	103	107
Induction [Fold]	0.9	1.0	1.0	1.0	1.0	1.1	1.2	-*	1.5	2.1	3.4
Standard Deviation	0.03	0.07	0.07	0.03	0.03	0.06	0.05	-*	0.18	0.32	0.32
p-value	-	-	-	-	-	-	0.42	-*	0.13	0.01	0.00

Results of Test Item Concentrations in Repetition I

Conc [µM]	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Precipi- tates	no	no	no	no	yes	yes	yes	yes	yes	yes	yes	yes
Viability [%]	130	119	123	1	1	4	3	4	3	4	1	0
Induction [Fold]	4.8	6.2	6.0	0.1	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
Standard Devia- tion	1.12	1.22	1.51	0.10	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
p-value	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00

Results of Controls in Repetition IV

	SC	SC	SC	SC	SC	SC	PC 4 µM	PC 8 µM	PC 16 µM	PC 32 µM	PC 64 µM
Viability [%]	91	88	101	108	105	107	105	110	109	105	102
Induction [Fold]	1.0	1.0	1.0	1.0	1.0	1.0	1.1	1.3	1.4	1.8	2.9
Standard Deviation	0.01	0.08	0.03	0.04	0.02	0.02	0.06	0.07	0.06	0.04	0.08
p-value	-	-	-	-	-	-	0.40	0.10	0.04	0.01	0.00

Results of Test Item Concentrations in Repetition IV

Conc [µM]	0.02	0.03	0.06	0.12	0.24	0.49	0.98	1.95	3.91	7.81	15.63	31.25
Precipi- tates	no	no	no	no	no	no	no	no	no	no	yes	yes
Viability [%]	92	103	109	115	115	111	126	125	127	129	-5	-3
Induction [Fold]	1.0	1.2	1.2	1.4	1.9	2.8	3.9	5.1	5.5	11.7	0.0	0.0
Standard Devia- tion	0.25	0.28	0.20	0.24	0.31	0.28	0.41	0.70	1.28	1.47	0.00	0.00
p-value	0.89	0.19	0.12	0.02	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00

Results of Controls in Repetition V

	SC	SC	SC	SC	SC	SC	PC 4 µM	PC 8 µM	PC 16 µM	PC 32 µM	PC 64 µM
Viability [%]	80	73	106	107	115	119	118	120	119	122	123
Induction [Fold]	0.9	1.0	1.0	1.0	1.1	1.1	1.2	1.2	1.3	1.5	2.1
Standard Deviation	0.02	0.08	0.01	0.01	0.03	0.04	0.04	0.01	0.06	0.12	0.15
p-value	-	-	-	-	-	-	0.02	0.01	0.00	0.00	0.00

Results of Test Item Concentrations in Repetition V

Conc [µM]	0.02	0.03	0.06	0.12	0.24	0.49	0.98	1.95	3.91	7.81	15.63	31.25
Precipi- tates	no	no	no	no	no	no	no	no	no	no	yes	yes
Viability [%]	82	95	114	128	138	144	137	153	138	0	1	1
Induction [Fold]	0.9	1.0	1.2	1.4	1.8	2.4	3.4	3.9	4.0	6.2	0.0	0.0
Standard Deviation	0.06	0.01	0.06	0.07	0.10	0.16	0.30	0.45	0.39	9.45	0.00	0.00
p-value	0.30	0.39	0.01	0.00	0.00	0.00	0.00	0.00	0.00	0.39	0.00	0.00

 

In repetition I cytotoxic effects were observed at the test item concentrations 2000 µM to 7.81 µM. For that reason, the concentrations for the following repetitions were adapted. In repetition IV a cytotoxic effect was observed at the two highest test item concentrations 31.25 µM and 15.63 µM. Against the expectations, no cytotoxic effect was detected at the concentration 7.81 µM. In repetition V cytotoxicity was observed at the test item concentrations 31.25 µM, 15.63 µM and 7.81 µM. All those concentrations were excluded from the evaluation of the luciferase induction. In addition, also the test item concentration 7.81 µM was not used for the assessment of the test item, since due to the extreme fluctuations of the results at this concentration in viability but also in luciferase induction and the knowledge that precipitates of the test were clearly visible at all higher concentrations, the large difference between the values strongly indicates that precipitates may also be present at this concentration, but are no longer visible to the naked eye even by microscopic analysis. For that reason, the results of this test item concentration were also not used for the assessment of the test item.

Finally, the following test item concentrations showed a viability ≥ 70 % and could therefore be evaluated for luciferase induction:

Repetition I: 0.98 µM, 1.95 µM, 3.91 µM

Repetition IV and V: 0.02 µM, 0.03 µM, 0.06 µM, 0.12 µM, 0.24 µM, 0.49 µM, 0.98 µM, 1.95 µM, 3.91 µM

In repetition I a statistically significant increase in luciferase induction ≥ 1.5 fold was measured at all evaluable concentrations (0.98 µM, 1.95 µM, 3.91 µM). But since no dose-dependent effect was observed the result of this repetition was considered as “inconclusive” according to the criteria of OECD 442D.

In repetition IV and V a statistically significant and reproducible dose-dependent increase in luciferase induction ≥ 1.5 fold was measured in all evaluable concentrations down to 0.24 µM. For that reason, the result of these two repetitions was considered as “positive” according to the criteria of OECD 442D.

The following values were calculated as the final results:

Imax: 7.9 fold (average of the two values of repetition IV and V)

EC1.5: 0.2 µM (geometric mean of the two values of repetition IV and V) IC50: 7.9 µM (geometric mean of the three values of repetition I, IV and V) IC30: 7.2 µM (geometric mean of the three values of repetition I, IV and V)

Repetition I has to be declared as “inconclusive” due to the missing dose-response and the results of repetition IV and V are considered as “positive”.

Applicant's summary and conclusion

Interpretation of results:

other: Classified as a Skin sensitiser in Category 1 as per the CLP Regulation EC No. 1272/2008

Conclusions:

It can be stated that under the experimental conditions of this study, the test item, ZnDBM - Dibenzoylmethanate zinc salt, was positive in the KeratinoSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor.

Executive summary:

The in vitro study was performed to investigate the potential of ZnDBM - Dibenzoylmethanate zinc salt to activate the Nrf2 transcription factor, by using the KeratinoSens cell line.

In total five repetitions (I, II, III, IV and V) were performed of which repetition II and III were invalid (standard deviation of solvent control was > 20 %) and had to be repeated. In the end three valid and independent repetitions (I, IV and V) were performed and evaluated.

None of the real treatment concentrations in all repetitions deviated more than 10 % from the nominal concentration.

In addition, the positive control cinnamic aldehyde was tested in a series of 5 concentrations ranging from 4 to 64 µM and fulfils all acceptability criteria.

In repetition I cytotoxic effects were observed at the test item concentrations 2000 µM to 7.81 µM. For that reason, the concentrations for the following repetitions were adapted. In repetition IV a cytotoxic effect was observed at the two highest test item concentrations 31.25 µM and 15.63 µM. Against the expectations, no cytotoxic effect was detected at the concentration 7.81 µM. In repetition V cytotoxicity was observed at the test item concentra- tions 31.25 µM, 15.63 µM and 7.81 µM. All those concentrations were excluded from the evaluation of the luciferase induction. In addition, also the test item concentration 7.81 µM was not used for the assessment of the test item, since due to the extreme fluctuations of the results at this concentration in viability but also in luciferase induction and the knowledge that precipitates of the test were clearly visible at all higher concentrations, the large difference between the values strongly indicates that precipitates may also be present at this concentration, but are no longer visible to the naked eye even by microscopic analysis. For that reason, the results of this test item concentration were also not used for the assessment of the test item.

In repetition I a statistically significant increase in luciferase induction ≥ 1.5 fold was measured at all evaluable concentrations (0.98 µM, 1.95 µM, 3.91 µM). But since no dose-dependent effect was observed the result of this repetition was considered as “inconclusive” according to the criteria of OECD 442D.

In repetition IV and V a statistically significant and reproducible dose-dependent increase in luciferase induction ≥ 1.5 fold was measured in all evaluable concentrations down to 0.24 µM. For that reason, the result of these two repetitions was considered as “positive” according to the criteria of OECD 442D.

As an additional information, a consideration of the test item in accordance to OECD 497 was also performed. Regarding this guideline, the results are identical to OECD 442D: Repetition I has to be declared as “inconclusive” due to the missing dose-response and the results of repetition IV and V are considered as “positive”.

In conclusion, it can be stated that under the experimental conditions of this study, the test item, ZnDBM - Dibenzoylmethanate zinc salt, was positive in the KeratinoSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor.