Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

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).

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.