Zinc acrylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 23, 2018 to February 02, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU-Method B.60 of the Commission Regulation (EU) No. 2017/735 adopted 14. Feb. 2017: “In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method”

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

Zinc acrylate

EC Number:

238-692-3

EC Name:

Zinc acrylate

Cas Number:

14643-87-9

Molecular formula:

C3H4O2.1/2Zn

IUPAC Name:

zinc acrylate

Test material form:

solid: particulate/powder

Details on test material:

Test Substance Name: Zinc acrylate
Batch no: SC1703131C
Test Facility Identification Number: 17102308G
Appearance: White powder
Composition: 99.5% Zinc acrylate
Purity: 99.5% Zinc acrylate
Homogeneity: Homogeneous
Expiry date: Sep. 09, 2018
Storage: Room emperature: (20 ± 5°C), keep away from humidity.

In vitro test system

Details on the study design:

Preparation
The solubility of the test substance was determined in a non-GLP pre-test in dimethyl sulfoxide (DMSO) and cell culture medium (DMEM) at 200mM. The test itemsubstance was insoluble in DMEM but nearly completely soluble in DMSO (stable suspension)at the required concentration (200 mM). Therefore, DMSO was used as solvent. Since the final concentration of the solvent during treatment is limited to 1 %, a stock solution containing 200 mM (CRFT) and 25 mM (experiment I and II) test itemsubstance in DMSO was pre-pared. Subsequent dilution to 1 % finally yielded a maximum concentration of 2000 µM in the pre-test and 250 µM in the 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 concentra-tions 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 corre-sponding 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 solutions as well as the dilutions were freshly prepared on the day of treatment.

Controls
Negative Control: Name: DL-Lactic acid, CAS no.: 50-21-5, Solvent: DMSO, Supplier: Sigma-Aldrich, Purity: 90.5 %, Lot no.: BCBP5043V, Expiry Date: 26. Jan. 2021, Final concentration: 5000 µM.
Positive Control: Name: EGDMA (Ethylene glycol dimethylacrylate), CAS no.: 97-90-5, Solvent: DMSO, Supplier: Sigma-Aldrich, Purity: 98 %, Lot no.: SHBG0572V, Expiry Date: 27. Jan. 2021, Final concentration: 120 µM.
Solvent Control: Name: DMSO, CAS no.: 67-68-5, Supplier: Carl Roth, Purity: 99.5 %, Lot no.: 187256959, Expiry Date: 04. Apr. 2020, Final concentration: 1 %
The solutions were freshly prepared on the day of the experiment.

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 (Ludwigs-hafen, 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 nitro-gen in the cell bank of LAUS GmbH to allow a continuous stock of cells, which guaran-tees similar parameters of the experiment and reproducible characteristics of the cells.
For the Cytotoxicity Range Finder Assay cells of passage 6 were used. For both main ex-periments cells of passage 8 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.

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 la-boratory material were also used.

Demonstration of proficiency
Prior to routine use, the validity of the LuSens test at LAUS GmbH was demonstrated in a proficiency study. In this study, 22 proficiency chemicals (indicated by the OECD 442D guideline as well as the OECD PERFORMANCE STANDARDS FOR ASSESSMENT OF PROPOSED SIMILAR OR MODIFIED IN VITRO SKIN SENSITISATION ARE-NRF2 LU-CIFERASE TEST METHODS) were tested. As prescribed by the guidelines, more than 80 % (96 %) of the results were correctly categorized. Therefore, the proficiency of the LuSens test was demonstrated.
For all control substances historical data are available (chapter 18, page 40), which demonstrates the reliability and the validity of those substances.

Results and discussion

Positive control results:

EGDMA (120 µM) was used as positive control. The viability was above 70 % and a distinct increase in lu-ciferase induction above 2.5 fold in comparison to the solvent control was detected. This luciferase induc-tion is well within the historical data range of the positive control.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Test substance concentration in Exp 1 : 33.6 µM, 40.4 µM, 48.5 µM, 58.1 µM, 69.8 µM, 83.7 µM, 100.5 µM, 120.6 µM

Parameter:

other: Minimum non-cytotoxic (Relative viability ≥70%) test substance concentration causing ≥ 1.5 fold luciferase induction (µM)

Value:

69.8

Vehicle controls validity:

valid

Remarks:

<1.5 fold luciferase induction

Negative controls validity:

valid

Remarks:

<1.5 fold luciferase induction in comparison to solvent control

Positive controls validity:

valid

Remarks:

2.5 fold luciferase induction in comparison to solvent control

Remarks on result:

positive indication of skin sensitisation

Results 2

Key result

Run / experiment:

other: Test substance concentration in Exp 1 : 33.6 µM, 40.4 µM, 48.5 µM, 58.1 µM, 69.8 µM, 83.7 µM, 100.5 µM, 120.6 µM, 144.7 µM, 173.6 µM

Parameter:

other: Minimum non-cytotoxic (Relative viability ≥70%) test substance concentration causing ≥ 1.5 fold luciferase induction (µM)

Value:

69.8

Vehicle controls validity:

valid

Remarks:

<1.5 fold luciferase induction in comparison to solvent control

Negative controls validity:

valid

Remarks:

<1.5 fold luciferase induction in comparison to solvent control

Positive controls validity:

valid

Remarks:

2.5 fold luciferase induction in comparison to solvent control

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

DL-lactic acid (5000 µM) was used as negative control. The viability was above 70% and the induction of the luciferase was <1.5 fold in comparison to the solvent control and well within the historical data range of the negative control. The induction of the luciferase of the growth control (Medium no. 3) was <1.5 fold. Since all acceptability criteria of the assay were met the study is valid.

Any other information on results incl. tables

Results

Results of Experiment I

The results of experiment I are indicated in table 8-a and figures 8-a and 8-b. 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: 0.9 fold, negative control: 1.1 fold). However, the positive control induced a clear effect with an induction value of 4.2 fold in comparison to the solvent control. No cytotoxic effect was observed at the test substance concentrations 33.6 µM to 120.6 µM. The viability values were all > 103 % and therefore analysable for luciferase induction. A cytotoxic effect was observed at the concentrations 144.7 µM to 250 µM. Therefore, the results of these concentrations were not included for the final evaluation. In the Luciferase assay, an induction of the luciferase above the threshold of 1.5 fold in comparison to the solvent control was detected at the concentrations 69.8 µM, 83.7 µM, 100.5 µM and 120.6 µM.

Table 8a Results of experiment 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	0.11	10.57	100.0	4.20	4.20
Growth Control	-	0.9	0.06	6.81	136.8	6.76	4.94
Negative Control	5000	1.1	0.06	6.04	110.2	5.71	5.19
Positive Control	120	4.2	0.22	5.23	96.4	2.01	2.09
Test substance	33.6	1.3	0.06	4.53	107.5	6.59	6.13
Test substance	40.4	1.3	0.12	9.49	103.5	3.07	2.96
Test substance	48.5	1.4	0.08	5.66	109.7	6.77	6.17
Test substance	58.1	1.4	0.03	2.04	104.9	1.02	0.98
Test substance	69.8	1.6	0.15	9.57	113.1	7.11	6.29
Test substance	83.7	2.1	0.14	6.64	111.5	7.20	6.46
Test substance	100.5	2.8	0.21	7.67	105.8	4.66	4.41
Test substance	120.6	4.5	0.24	5.31	104.5	7.52	7.20
Test substance	144.7	6.7*	0.91	13.69	67.9	2.75	4.05
Test substance	173.6	4.4*	0.91	20.53	20.9	7.18	34.30
Test substance	208.3	1.1*	0.06	5.26	2.4	1.11	46.27
Test substance	250.0	0.2*	0.11	54.54	1.4	1.80	133.23

* = Due to cytotoxicity, these values were not used for the final evaluation of luciferase induction.

Graphical presentation of results of tested test substance and control substances concentrations are presented in figure 8a and figure 8b, attached document in background material section.

Results of Experiment II

The results of experiment II are indicated in table 8-b and figures 8-c and 8-d. 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: 1.0 fold). However, the positive control induced a clear effect with an induction value of 5.1 fold in comparison to the solvent control. No cytotoxic effect was observed at the test substance concentrations 33.6 µM to 173.6 µM. The viability values were all ≥ 80 % and therefore analysable for luciferase induction. A cytotoxic effect was observed at the concentrations 208.3 µM and 250 µM. Therefore, the results of these concentrations were not included for the final evaluation. In the Luciferase assay, an induction of the luciferase above the threshold of 1.5 fold in comparison to the solvent control was detected at the concentrations 69.8 µM, 83.7 µM, 100.5 µM, 120.6 µM, 144.7 µM and 173.6 µM. 

Table 8b Results of experiment 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	0.09	8.96	100.0	4.26	4.26
Growth Control	-	1.0	0.07	7.19	139.7	4.28	3.06
Negative Control	5000	1.0	0.05	4.60	110.1	2.32	2.11
Positive Control	120	5.1	0.22	4.44	100.7	3.96	3.94
Test substance	33.6	1.4	0.11	7.77	110.8	1.50	1.35
Test substance	40.4	1.3	0.05	3.79	112.5	3.27	2.90
Test substance	48.5	1.4	0.12	8.76	111.2	2.20	1.98
Test substance	58.1	1.4	0.04	2.73	115.1	3.51	3.05
Test substance	69.8	1.7	0.07	4.11	112.2	3.92	3.49
Test substance	83.7	1.8	0.08	4.25	112.5	3.32	2.95
Test substance	100.5	2.5	0.12	4.87	112.0	3.79	3.39
Test substance	120.6	3.9	0.05	1.34	116.0	3.28	2.83
Test substance	144.7	6.0	0.21	3.54	105.4	1.72	1.63
Test substance	173.6	10.0	0.55	5.48	80.0	2.16	2.70
Test substance	208.3	6.6*	0.50	7.62	18.4	5.03	27.43
Test substance	250.0	1.3*	0.43	32.19	3.3	0.46	14.14

* = Due to cytotoxicity, these values were not used for the final evaluation of luciferase induction.

Graphical presentation of results of tested test substance and control substances concentrations are presented in figure 8c and figure 8d, attached document in background material section.

Evaluation

Acceptability

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

Table 9a Acceptability of experiment I and II

Criteria	Found in experiment I	Found in experiment 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: 4.2 Relative viability: 96.4 %	Positive control Fold induction: 5.1 Relative viability: 100.7 %
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: 1.1 Relative viability: 110.2 % Growth control: Fold induction: 0.9 Relative viability: 136.8 %	Negative control: Fold induction: 1.0 Relative viability: 110.1 % Growth control: Fold induction: 1.0 Relative viability: 139.7 %
The average percentage standard deviation (luciferase induction) of the variability in at least 21 solvent control wells should be below 20 %.	10.57 %	8.96 %
At least 3 test concentrations must be within viability limits, i.e. have relative viability of at least 70 %.	8 concentrations are analysable	10 concentrations are analysable

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

Classification

Each valid experiment (i.e. meeting all acceptance criteria, according to the procedure described above) is interpreted as follows:

1) A test compound is considered to have the potential to activate the Nrf2 transcription factor if 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 experiments.

 

2) A test compound is considered not to have the potential to activate the Nrf2 transcription factor if the effects mentioned above are not observed.

3) 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 on the skin sensitization hazard of a substance, a minimum of two valid and independent experiments needs to indicate a positive or negative result according to the above-described criteria. If the first two experiments come to the same result (i.e. either being negative or being positive) no further testing is required. In case that the first two experiments give discordant results (i.e. one is negative and the other is positive), a third independent experiment needs to be conducted to complete the study. The skin sensitizing potential (corresponding to 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 experiments are negative/positive, the substance is considered as negative/positive. The luciferase induction was above 1.5 fold in more than 2 consecutive non-cytotoxic test substance concentrations in experiment I and II. Therefore, the test substance Zinc acrylate is considered to have the potential to activate the Nrf2 transcription factor (sensitizing potential) under the conditions of the LuSens test.

Discussion and Conclusion

This in vitro study was performed to investigate the potential of Zinc acrylate to activate the Nrf2 transcription factor (sensitizing potential), by using the LuSens cell line. The assay was performed in two independent experiments. 12 concentrations of the test substance were evaluated. The exposure time was 48 h. The following nominal concentrations of the test substance were investigated in experiment I and II: 33.6 µM, 40.4 µM, 48.5 µM, 58.1 µM, 69.8 µM, 83.7 µM, 100.5 µM, 120.6 µM, 144.7 µM, 173.6 µM, 208.3 µM, 250.0 µM. None of the real treatment concentrations in both experiments deviated more than 10 % from the nominal concentration. Precipitation of the test substance was not visible up to the highest concentration.

EGDMA (120 µM) was used as positive control. The viability was above 70 % and a distinct increase in luciferase induction above 2.5 fold in comparison to the solvent control was detected. This luciferase induction is well within the historical data range of the positive control. DL-lactic acid (5000 µM) was used as negative control. The viability was above 70 % and the induction of the luciferase was < 1.5 fold in comparison to the solvent control and well within the historical data range of the negative control.

The induction of the luciferase of the growth control (Medium no. 3) was < 1.5 fold. Since all acceptability criteria of the assay were metthe study is valid. In experiment I a cytotoxic effect was observed at the concentrations 144.7 µM to 250 µM.

In experiment II a cytotoxic effect was observed at the concentrations 208.3 µM and 250 µM. Finally the following test substance concentrations showed a viability ≥ 70 % and could therefore be evaluated for luciferase induction:

Experiment I: 33.6 µM, 40.4 µM, 48.5 µM, 58.1 µM, 69.8 µM, 83.7 µM, 100.5 µM, 120.6 µM

Experiment II: 33.6 µM, 40.4 µM, 48.5 µM, 58.1 µM, 69.8 µM, 83.7 µM, 100.5 µM, 120.6 µM, 144.7 µM, 173.6 µM

A substantial and reproducible dose-dependent and statistically significant increase in luciferase induction was measured in the test substance concentrations 69.8 µM, 83.7 µM, 100.5 µM and 120.6 µM in experiment I and in the test substance concentrations 69.8 µM, 83.7 µM, 100.5 µM, 120.6 µM, 144.7 µM and 173.6 µM in experiment II.

In conclusion, it can be stated that under the experimental conditions of this study, the test substance, Zinc acrylate, was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor (sensitizing potential).

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Under the study conditions, the test substance was concluded to be a skin sensitiser.

Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance using the ARE-Nrf2 Luciferase test method, according to OECD Guideline 442D and EU Method B.60, in compliance with GLP. Two independent experiments were performed. In these experiments, cells were incubated with test substance at 12 different concentrations in range of 33.6 - 250.0 µM for 48 h. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control to determine test substance concentration needed for a statistically significant induction of luciferase activity above the threshold (i.e. EC1.5 value). In addition, the viability was assessed with an MTT assay. EC1.5 value observed with at least three non-cytotoxic tested test substance concentrations (Cell viability of >70%) compared to the vehicle control was considered positive for skin sensitisation. Following test substance concentrations showed a viability ≥70% and could therefore be evaluated for luciferase induction: Experiment I: 33.6, 40.4, 48.5, 58.1, 69.8, 83.7, 100.5, and 120.6 µM; Experiment II: 33.6, 40.4, 48.5, 58.1, 69.8, 83.7, 100.5, 120.6, 144.7, and 173.6 µM. A substantial and reproducible dose-dependent and statistically significant (p <0.05) increase in luciferase induction (EC ≥1.5) was measured at the test substance concentrations 69.8 µM and above in both experiment I and experiment II. All control substances indicated the expected effect, validating the study. The positive control (ethylene glycol dimethylacrylate) induced a clear effect with an induction value of 4.2 -5.1 fold in comparison to the solvent control. Since positive results (>1.5 fold induction) were observed at test substance concentrations with a cell viability of >70% compared to the vehicle control in both experiments, it can be stated that the test substance was positive in the LuSens assay. The study met the validity criteria. Under the study conditions, the test substance was concluded to be a skin sensitiser (Frühmesser, 2018).