Zinc acrylate

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

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.

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.

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

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.

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).

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).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 07, 2018 to February 13, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)

Version / remarks:

OECD TG 442E: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), adopted 29 Jul 2016.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Details on the study design:

Reasons for the choice of the THP-1 cell Line: The OECD 442E indicates that the human monocytic leukaemia cell line, THP-1 should be used for the h-CLAT. The cells were purchased by CLS (Eppelheim, Germany).
Cell cultures: THP-1 cells are stored in liquid nitrogen in the cell bank of LAUS GmbH to allow a con-tinuous stock of cells, which guarantees similar parameters of the experiment and repro-ducible characteristics of the cells. The THP-1 cells are routinely seeded every 2-3 days at the density of 0.1 – 0.2 * 106 cells/mL. They were maintained at densities from 0.1 to 1.0 * 106 cells/mL. Prior to using them for testing, the cells were qualified by conducting a reactivity check. For the pre-test cells of passage 12 were used. For the main experiments cells of passage 14 were used. After thawing the cells were cultivated in RPMI 1640 complete culture me-dium in cell culture flasks at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.

Reactivity check: Three weeks after thawing, a reactivity check of the cells was performed. For that, the two positive controls 2,4-dinitrochlorobenzene (DNCB) (CAS n. 97-00-7, ≥ 99% purity, test concentration: 4 µg/mL) and nickel sulfate (NiSO4) (CAS n. 10101-97-0, ≥ 99% purity, test concentration: 200 µg/mL) as well as the negative control, lactic acid (LA) (CAS n. 50-21-5, ≥ 85% purity, test concentration: 1000 µg/mL) were used. These substances as well as all additional information are given by the OECD 442E. The experimental procedure was identical to the experiments in this study. The two positive controls produced a clearly positive result of the two surface markers CD86 and CD54. The negative control produced a clearly negative result of both surface markers. Therefore, the cells were found to be suitable for the experiments. For the pre-test as well as the experiments, only cells which have successfully passed the reactivity check were used.

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 on 96- and 24-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.

Demonstration of proficiency: Prior to routine use, the validity of the h-CLAT test at LAUS GmbH was demonstrated in a proficiency study. In this study, 10 proficiency chemicals (indicated by the OECD 442E guideline) were tested. As prescribed by the guideline, more than 7 of the results were correctly categorized. Therefore, the proficiency of the test system was demonstrated. For all control substances historical data are available, which demonstrate the reliability and the validity of those substances.
Prior to use in the pre-test and the experiments, the proficiency of the cells was demonstrated in a reactivity check. Only the cells which passed the reactivity check were used for the pre-test and the experiments. The runs for Experiment I and II were performed on the same day, provided that for each run: a) independent fresh stock solutions and working solutions of the test substance and antibody solutions were pre-pared and b) independently harvested cells were used (cells came from the same pas-sage and were collected from different culture flasks.)

Positive control results:

The two positive controls produced a clearly positive result of the two surface markers CD86 and CD54. The negative control produced a clearly negative result of both surface markers. Therefore, the cells were found to be suitable for the experiments.

Key result

Run / experiment:

other: Average of two run

Parameter:

other: CV75 (µg/mL)

Remarks:

A concentration showing 75% of THP-1 cell survival (25% cytotoxicity)

Value:

157.26

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment I

Parameter:

other: CD54 expression measured as RFI (76.0 µg/mL to 189.0 µg/mL)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: Experiment II

Parameter:

other: CD54 expression measured as RFI (109.4 µg/mL to 189.0 µg/mL)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: Experiment I

Parameter:

other: CD86 expression measured as RFI (52.7 µg/mL and 63.3 µg/mL as well as at the concentrations 91.1 µg/mL to 189.0 µg/mL)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: Experiment II

Parameter:

other: CD86 expression measured as RFI (131.3 µg/mL and 157.5 µg/mL)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

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

Results of the pre-test

Substance	Concentration	Viability
	[µg/mL]	[%]
Medium	-	88.51
Solvent control test substance (DMSO)	-	88.85
Solvent control positive control (DMSO)	-	89.07
Positive control DNCB	4.0	84.89
Test substance	7.8	89.26
Test substance	15.6	89.46
Test substance	31.3	89.33
Test substance	62.5	90.00
Test substance	125.0	86.54
Test substance	250.0	51.70
Test substance	500.0	4.09
Test substance	1000.0	2.04

Results from experiment I

	Concen-tration	Viability	Antibodies	MFI Value	MFI ratioto Isotype	RFI Value
	[µg/mL]				[%]	[%]
Medium	-	95.69%	CD86	1438	164
		95.70%	CD54	1152	132
		95.69%	ISO	875
DMSO	-	96.61%	CD86	1201	139	60
		96.14%	CD54	1164	135	108
		96.27%	ISO	864
DMSO	-	96.53%	CD86	1278	146	72
		96.51%	CD54	1263	145	140
		95.95%	ISO	874	-
DNCB	4.0	85.10%	CD86	3007	-	505
		81.34%	CD54	2158	-	306
		81.06%	ISO	966	-
Test Substance	52.7	92.53%	CD86	1541	-	203
		93.04%	CD54	1331	-	158
		92.89%	ISO	857	-
Test Substance	63.3	91.84%	CD86	1416	-	160
		91.58%	CD54	1425	-	182
		91.86%	ISO	878	-
Test Substance	76.0	91.47%	CD86	1313	-	125
		91.08%	CD54	1514	-	208
		90.03%	ISO	891	-
Test Substance	91.1	88.71%	CD86	1518	-	206
		88.05%	CD54	1835	-	337
		86.84%	ISO	825	-
Test Substance	109.4	86.14%	CD86	2025	-	304
		86.77%	CD54	2516	-	505
		85.98%	ISO	1000	-
Test Substance	131.3	81.53%	CD86	1866	-	237
		79.70%	CD54	3070	-	668
		79.55%	ISO	1067	-
Test Substance	157.5	76.02%	CD86	1715	-	187
		75.55%	CD54	3106	-	674
		76.69%	ISO	1085	-
Test Substance	189.0	72.46%	CD86	2010	-	223
		75.37%	CD54	2826	-	522
		77.69%	ISO	1259	-

Calculated EC200 (for CD54): 72.09 µg/mL

Results from experiment II

	Concen-tration	Viability	Antibodies	MFI Value	MFI ratioto Isotype	RFI Value
	[µg/mL]				[%]	[%]
Medium	-	96.32%	CD86	1664	197
		95.56%	CD54	1174	139
		95.75%	ISO	845
DMSO	-	95.09%	CD86	1568	188	89
		95.72%	CD54	1233	148	121
		95.66%	ISO	835
DMSO	-	95.55%	CD86	1414	175	74
		96.15%	CD54	1196	148	117
		95.71%	ISO	810
DNCB	4.0	86.66%	CD86	3667		450
		84.27%	CD54	2162		315
		83.64%	ISO	947
Test Substance	52.7	94.22%	CD86	1632		110
		92.81%	CD54	1255		108
		94.06%	ISO	826
Test Substance	63.3	92.77%	CD86	1482		89
		92.23%	CD54	1375		137
		92.69%	ISO	831
Test Substance	76.0	91.07%	CD86	1451		85
		90.63%	CD54	1532		177
		90.74%	ISO	826
Test Substance	91.1	90.71%	CD86	1466		80
		92.37%	CD54	1522		162
		92.33%	ISO	878
Test Substance	109.4	87.97%	CD86	1939		138
		88.96%	CD54	1870		238
		88.30%	ISO	924
Test Substance	131.3	84.64%	CD86	2377		188
		84.42%	CD54	2316		332
		83.87%	ISO	996
Test Substance	157.5	77.39%	CD86	2252		153
		77.80%	CD54	2959		460
		79.37%	ISO	1129
Test Substance	189.0	73.54%	CD86	2129		127
		75.23%	CD54	3338		537
		79.59%	ISO	1200

 Calculated EC200 (for CD54): 100.25 µg/mL

Classification

For CD86/CD54 expression measurement, each test substance is tested in at least two independent runs to derive a single prediction. A h-CLAT prediction is considered positive if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs, otherwise the h-CLAT prediction is considered negative:

− The RFI of CD86 is ≥ than 150 % at any tested concentration with cell viability ≥ 50 %

− The RFI of CD54 is ≥ than 200 % at any tested concentration with cell viability ≥ 50 %

If the first two runs are concordant for both markers, a third experiment is not necessary. Then, the study is completed. If the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 and the other is only positive for CD54, a third run is required. If this third run is negative for both markers, the h-CLAT prediction is considered negative. On the other hand, if the third run is positive for either marker or for both markers, the h-CLAT prediction is consid-ered positive.

In case of a negative result, special care should be taken if the test substance

1) has a Log Kow > 3.5. Those results should not be considered. However, positive results obtained with those test chemicals can be used for analysis.

2) is a pro-hapten or a pre-hapten

3) has an autofluorescence and is emitting at the same wavelength as FITC or as PI

 In accordance to these classification criteria the test substance test substance was positive in the h-CLAT and is therefore considered to have the potential to activate dendritic cells and therefore to be a potential skin sensitizer.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Under 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 human Cell Line Activation Test (h-CLAT) method according to OECD Guideline 442E, in compliance with GLP. The h-CLAT assay measures markers of dendritic cell (DC) activation in the human leukaemia cell line THP-1. The ability of a chemical to induce activation of CD86 and CD54 in THP-1 cells was used as an assessment of the chemical’s skin sensitisation potential. Activation of CD54 and CD86 protein markers was measured by flow cytometry following 24 h exposure to the test substance. The concentration of the test substance used was selected on the basis of a pre-determined CV75 value (i.e. the estimated concentration yielding 75% cell viability). For the CV75 determination, THP-1 cells (a cell line that mimics DCs) were pre-cultured for either 48 or 72 h. Following this, the cells were dosed with the test substance over an 8 dose range (7.8, 15.6, 31.3, 62.2, 125, 250, 500 and 1000 µg/mL) and incubated for 24 ±0.5 h. The cells were then washed and stained with propidium iodide which allows for discrimination of live/dead cells by flow cytometry. The CV75 was found to be157.26 µg/mL. In experiment I the Relative fluorescence intensity (RFI) of CD86 was ≥150% at the test substance concentrations 52.7 µg/mL and 63.3 µg/mL as well as at the concentrations 91.1 µg/mL to 189.0 µg/mL. The RFI of CD54 was ≥ 200% at the test substance concentrations 76.0 µg/mL to 189.0 µg/mL. In this experiment, no EC150 could be determined, because the positive values were not continuous. EC200 was 72.09 µg/mL. In experiment II the RFI of CD86 was ≥ 150 % at the test substance concentrations 131.3 µg/mL and 157.5 µg/mL. The RFI of CD54 was ≥ 200% at the test substance concentrations 109.4 µg/mL to 189.0 µg/mL. Again, no EC 150 could be determined. EC200 was 100.25 µg/mL. As the CD54/CD86 expression crossed the sensitisation threshold the test substance was classified as a skin sensitiser. Experiment I and II all of the tested concentrations showed viability values above 50%. Acceptance criteria for all controls and the test substance were met in both runs for the CV75 determination and for the measurement of CD54 and CD86 expression. Under study conditions, the test substance was concluded to be a skin sensitiser (Frühmesser, 2018).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Zinc diacrylate studies

Study 1:

A study was conducted to determine the skin sensitisation potential of the test substance using the human Cell Line Activation Test (h-CLAT) method according to OECD Guideline 442E, in compliance with GLP. The h-CLAT assay measures markers of dendritic cell (DC) activation in the human leukaemia cell line THP-1. The ability of a chemical to induce activation of CD86 and CD54 in THP-1 cells was used as an assessment of the chemical’s skin sensitisation potential. Activation of CD54 and CD86 protein markers was measured by flow cytometry following 24 h exposure to the test substance. The concentration of the test substance used was selected on the basis of a pre-determined CV75 value (i.e. the estimated concentration yielding 75% cell viability). For the CV75 determination, THP-1 cells (a cell line that mimics DCs) were pre-cultured for either 48 or 72 h. Following this, the cells were dosed with the test substance over an 8 dose range (7.8, 15.6, 31.3, 62.2, 125, 250, 500 and 1000 µg/mL) and incubated for 24 ±0.5 h. The cells were then washed and stained with propidium iodide which allows for discrimination of live/dead cells by flow cytometry. The CV75 was found to be157.26 µg/mL. In experiment I the Relative fluorescence intensity (RFI) of CD86 was ≥150% at the test substance concentrations 52.7 µg/mL and 63.3 µg/mL as well as at the concentrations 91.1 µg/mL to 189.0 µg/mL. The RFI of CD54 was ≥ 200% at the test substance concentrations 76.0 µg/mL to 189.0 µg/mL. In this experiment, no EC150 could be determined, because the positive values were not continuous. EC200 was 72.09 µg/mL. In experiment II the RFI of CD86 was ≥ 150 % at the test substance concentrations 131.3 µg/mL and 157.5 µg/mL. The RFI of CD54 was ≥ 200% at the test substance concentrations 109.4 µg/mL to 189.0 µg/mL. Again, no EC 150 could be determined. EC200 was 100.25 µg/mL. As the CD54/CD86 expression crossed the sensitisation threshold the test substance was classified as a skin sensitiser. Experiment I and II all of the tested concentrations showed viability values above 50%. Acceptance criteria for all controls and the test substance were met in both runs for the CV75 determination and for the measurement of CD54 and CD86 expression. Under study conditions, the test substance was concluded to be a skin sensitiser (Frühmesser, 2018).

Study 2:

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).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of in vitro hCLAT and LuSens assays, the test substance was concluded to be a skin sensitiser, requiring classification as Skin Sens. 1 - H317: May cause an allergic skin reaction according to EU CLP (EC 1272/2008) criteria.