Registration Dossier

Administrative data

Description of key information

OECD Guideline 442E – Annex II ‘In Vitro Skin Sensitisation: U937 Cell Line Activation Test (USENS ™): postive

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

Based on the results of the two in vitro assays, the substance was found to be sensitising to skin.

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:
weight of evidence
Study period:
2018-11-07 to 2019-01-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD 442E – Annex II ‘In Vitro Skin Sensitisation: U937 Cell Line Activation Test (USENS ™)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of dendritic cells
Details on study design:
Skin sensitisation (In vitro test system) - Details on study design:
The U-SENS™ method is proposed to address this third key event by quantifying the change in the expression of a cell surface marker associated with the process of activation of monocytes and DC (i.e. CD86), in the human histiocytic lymphoma cell line U937, following exposure to sensitisers. The measured expression levels of CD86 cell surface marker in the cell line U937 is then used for supporting the discrimination between skin sensitisers and nonsensitisers.

Test System: U937 human monocytes.
Justification: Inducible CD86-expressing cells
Source: ATCC (American Type Culture Collection, Virginia, USA). ATCC no.: CRL-1593.2TM.

Experimental design:
Plating of Cells
Cultures were initiated in 96-well plates using 100 μL/well of a cell suspension adjusted at 5.0 x 105 viable cells/mL. Cell viability was > 90%. All assays were performed using two replicate culture-wells for the test item. One replicate was dedicated to the nonspecific IgG1 binding and the other one to the CD86 binding. Three replicates of untreated control (RPMI), vehicle control (in case of DMSO as vehicle), negative (LA) and positive (TNBS) controls were tested.
Three valid experiments were conducted per test item to demonstrate reproducibility of the results and conclusion. Initially, experiment 1 was rejected due to technical reasons and was repeated.

Treatment of Cells
Cells are treated for 45 ± 3 hours with the selected doses or controls (100 μL). The test item was in the first experiment evaluated up to 200 μg/mL using six doses: 1.0, 10, 20, 50, 100 and 200 μg/mL.
In the second and third experiments cells were treated with 8 selected doses of the test item. At least 2 concentrations were common with the previous experiment. The concentrations selected in the second and third experiment were as follows:
Second experiment: 90, 70, 50, 40, 30, 25, 20 and 15 μg/mL
Third experiment: 100, 90, 70, 50, 40, 30, 25 and 20 μg/mL
In all experiments, an untreated control (RPMI), vehicle control (in case of DMSO as vehicle) and the positive (TNBS) and negative control (LA) items were included. The final volume in the wells was 200 μL.
After 45 ± 3 hours of exposure, wells were checked for precipitate.

Cell antibodies staining for IgG1 and CD86
Cultures were transferred into V-shaped 96-well plates. The cells were separated from the exposure medium by centrifugation (5 min, 200 g). The supernatant was discarded and cells were rinsed once with 100 μL/well Phosphate Buffered Saline (PBS) containing 5% FCS. After a second centrifugation step (5 min, 200 g) 100 μL/well of the staining buffer (PBS containing 5% FCS) was applied to the cells.
FITC-conjugated antibodies was used for both IgG1 and CD86 staining:
- Mouse IgG1 of unknown specificity, for isotypic control (#555748; BD, Amsterdam, The Netherlands)
- Human CD86 specific mouse IgG1 (#555657; BD, Amsterdam, The Netherlands)
The cells were transferred into new V-shaped 96-well plates (keeping the same plate template) containing 5 μL/well of the appropriate antibody (1:1 diluted in PBS) and placed refrigerated in the dark for 30 minutes. After this staining period, the cells were rinsed twice with a mixture of PBS/FCS and once in PBS alone and re-suspended in 90 μL of PBS.

Analysis of the cells was performed by flow cytometry.

ACCEPTABILITY CRITERIA
- At the end of the incubation treatment period, the mean viability of the triplicate untreated (RPMI) U937 cells is > 90%
- The validity of the DMSO vehicle control is assessed by calculating a DMSO S.I. compared to untreated cells, and the mean viability of the triplicate cells is > 90%. The DMSO vehicle control is valid if the mean value of its triplicate CD86 S.I. was smaller than 250% of the mean of the triplicate CD86 S.I. of untreated U937 cells.
- The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and ≤ 25%.
- At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6% and < 1.5%.
- No drift in CD86 expression is observed. A drift is defined by i) the corrected %CD86+ value of the untreated control replicate 3 is less than 50% of the mean of the corrected %CD86+ value of untreated control replicates 1 and 2; and ii) the corrected
%CD86+ value of the negative control replicate 3 is less than 50% of mean of the corrected %CD86+ value of negative control replicates 1 and 2.
- The positive control (TNBS) is considered valid if at least two out of the three wells are positive (CD86 S.I. ≥ 150%) and non-cytotoxic (cell viability ≥ 70%).
- Negative control LA is considered valid if at least 2 out of 3 LA wells are negative (CD86-IgG1 SI < 150%) and non-cytotoxic (cell viability ≥ 70%).

ANALYSIS
CV70 calculations
When the CV70 cannot be calculated it is indicated as “NA” (Not Applicable). Otherwise the calculation is done as follows:
For each culture (IgG1 well and CD86 well), the percentage of viable cells (PI negative cells) was evaluated. The viability for each dose level is the mean of the IgG1 well and CD86 well. The theoretical concentration at which the chemical induces 30% cytotoxicity (i.e., 70% viability) was calculated.

EC150 calculations
When the EC150 cannot be calculated it is indicated as “NA” (Not Applicable). Otherwise the calculation was done as follows:
For each CD86 well culture, the percentage of induced CD86+ cells is calculated as: [absolute %CD86+ — absolute%IgG1+]
A stimulation index (S.I.) is calculated for each dose level.
The viability for each dose level are the mean of the IgG1 well and the CD86 well. The theoretical concentration at which the chemical induces a S.I. of 150 (i.e., 50% of CD86+ cells over the vehicle control) was calculated.

INTERPRETATION
- For CD86 expression measurement, each test chemical is tested in at least two independent runs (performed on a different day) to derive a single prediction (NEGATIVE or POSITIVE).
- The individual conclusion of an U-SENS™ run is considered Negative (hereinafter referred to as N) if the S.I. of CD86 is less than 150% at all non-cytotoxic concentrations (cell viability ≥ 70%) and if no interference (cytotoxicity, solubility or colour) is observed. Solubility interference is defined as crystals or drops observed under the microscope at 45 ± 3h post treatment (before the cell staining). Colour interference is defined as a shift of the FITC-labelled IgG1 dot-plot (IgG1 FL1 X Median S.I. ≥ 150%).
- In all other cases: S.I. of CD86 higher or equal to 150% and/or interferences observed, the individual conclusion of an U-SENS™ run is considered Positive (hereinafter referred to as P).
- An U-SENS™ prediction is considered NEGATIVE if at least two independent runs are negative (N) (Figure 1). If the first two runs are both negative (N), the U-SENS™ prediction is considered NEGATIVE and a third run does not need to be conducted.
- An U-SENS™ prediction is considered POSITIVE if at least two independent runs are positive (P) (Figure 1). If the first two runs are both positive (P), the U-SENS™ prediction is considered POSITIVE and a third run does not need to be conducted.
- There is an exception if, in the first run, the S.I. of CD86 is higher or equal to 150% at the highest non-cytotoxic concentration only. The run is then concluded NO CONCLUSION (NC), and additional concentrations (between the highest non cytotoxicity concentration and the lowest cytotoxicity concentration) should be tested in additional runs. A run NC conducts automatically to the need of at least 2 more runs, and to a fourth run in case runs 2 and 3 are not concordant (N and/or P independently). Follow up runs will be considered positive even if only one non cytotoxic concentration gives a CD86 equal or above 150%, since the dose setting has been adjusted for the specific test chemical. The final prediction will be based on the majority result of the three or four individual runs (i.e. 2 out of 3 or 2 out of 4).
Key result
Parameter:
other: S.I. of CD86 [%]
Run / experiment:
1
Value:
>= 150
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: In the first experiment, the S.I. of CD86 is higher or equal to 150% at the highest noncytotoxic concentration only (50 µg/mL). This run is therefore considered ‘No Conclusion’ and two subsequent experiments were performed.
Key result
Parameter:
other: S.I. of CD86 [%]
Run / experiment:
2
Value:
>= 150
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: the S.I. of CD86 is higher or equal to 150% at the highest noncytotoxic concentration
Key result
Parameter:
other: the S.I. of CD86 [%]
Run / experiment:
3
Value:
>= 150
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: the S.I. of CD86 is higher or equal to 150% at all tested concentrations
Other effects / acceptance of results:
All three experiments passed the acceptance criteria:
- At the end of the incubation treatment period, the mean viability of the triplicate untreated U937 cells was above the threshold of 90% (100%, 98% and 99% in experiment 1, 2 and 3, respectively).
- The mean viability of the triplicate DMSO vehicle control cells was above the threshold of 90% (100% in experiment 1 and 99% in experiment 2 and 3).
- The DMSO vehicle control mean value of its triplicate CD86 S.I. was smaller than 250% of the mean of the triplicate CD86 S.I. of untreated U937 cells in all three experiments.
- The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and ≤ 25% in all experiments.
- At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6% and < 1.5% in all experiments.
- No drift in CD86 expression was observed in the untreated controls (RPMI) and negative (LA) controls.
In all experiments the positive and negative control were considered valid. Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Experiment 1

-No precipitation was observed at the end of the incubation period in the 96-well plates.

-The test item showed toxicity, the calculated CV70 was 78 μg/mL.

-A biologically relevant increase in the expression of CD86 was observed after treatment with the test item, the EC150 is 22 μg/mL.

-The test item showed colour interference at 100 and 200 μg/mL.

-The positive control (TNBS) showed a S.I. ≥508% in all wells and was non-cytotoxic atall concentrations (cell viability ≥70%). The negative control (LA) showed a S.I.97% in all wells and was non-cytotoxic at all concentrations (cell viability ≥70%).

 

Experiment 2

-No precipitation was observed at the end of the incubation period in the 96-well plates.

-The test item showed toxicity, the calculated CV70 was 86 μg/mL.

-A biologically relevant increase in the expression of CD86 was observed after treatment with the test item, the EC150 is 63 μg/mL.

-The test item showed no colour interference.

-The positive control (TNBS) showed a S.I. ≥591% in all wells and was non-cytotoxic atall concentrations (cell viability ≥ 70%).The negative control (LA) showed a S.I.109% in all wells and was non-cytotoxic at all concentrations (cell viability≥ 70%).

 

Experiment 3

-No precipitation was observed at the end of the incubation period in the 96-well plates.

-The test item showed toxicity, the calculated CV70 was 61 μg/mL.

-A biologically relevant increase in the expression of CD86 was observed after treatment with the test item, the EC150 is < 20 μg/mL.

-The test item showed colour interference at 70 and 90 μg/mL.

- The positive control (TNBS) showed aS.I. ≥609% in all wells and was non-cytotoxic at all concentrations(cell viability ≥70%). The negative control (LA) showed a S.I.100% in all wells and was non-cytotoxic at all concentrations(cell viability ≥70%).

 

Table 1

Overview EC150 and CV70 Values

 

EC150 (μg/mL)

CV70 (μg/mL)

Test item Experiment 1

22

78

Test item Experiment 2 

63

86

Test item Experiment 3

< 20

61

 

Interpretation of results:
other: positive, to be used as part of WoE-conclusion
Conclusions:
The test substance showed a positive result (potential to increase the expression levels of CD86 cell surface marker in the U937 cell line)
Executive summary:

The objective of this study is to evaluate the ability of the test item to increase the expression levels of CD86 cell surface marker in the U937 cell line in the U937 cell line activation Test (U-Sens™) assay. The test item was dissolved in dimethyl sulfoxide at 50 mg/mL, 22.5 mg/mL and 25 mg/mL in experiment 1, 2 and 3, respectively. In the first experiment the stock was diluted to six test concentrations (1, 10, 20, 50, 100 and 200μg/mL). In the second and third experiment, a narrower dose-response analysis was performed to investigate the increase in expression of experiment 1 up to 90 and 100 μg/mL (experiment 2 and 3, respectively) in more detail. No precipitate was observed at any dose level tested. Three independent experiments were performed. In the first experiment the test item showed toxicity (CV70 value of 78 μg/mL) and a biologically relevant, induction of the CD86 activity (EC150 value of 22 μg/mL) was measured. The individual run conclusion is considered No Conclusion, since the S.I. of CD86 is higher or equal to 150% at the highest non-cytotoxic concentration only. In the second experiment the test item showed toxicity (CV70 value of 86 μg/mL) and a biologically relevant, induction of the CD86 activity (EC150 value of 63 μg/mL) was measured. The individual run conclusion is considered Positive. An additional third experiment was performed, since the first two experiments were not concordant. In the third experiment the test item showed toxicity (CV70 value of 61 μg/mL) and a biologically relevant, induction of the CD86 activity (EC150 value of < 20 μg/mL) was measured. The individual run conclusion is considered Positive. The test item is classified as Positive in the U-Sens™ assay since positive results (> 150% increase) were observed at test concentrations with a cell viability of >70% compared to the vehicle control. In conclusion, the test item is classified as positive (increase in the expression levels of CD86 cell surface marker in the U937 cell line) under the experimental conditions described.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2018-11-23 to 2019-01-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
June 2018
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Details on study design:
Skin sensitisation (In vitro test system) - Details on study design:
The existing knowledge of the chemical and biological mechanisms associated with skin sensitization has been summarized in the form of an Adverse Outcome Pathway (AOP). The second key event in this AOP takes place in the keratinocytes and includes inflammatory responses as well as gene expression associated with specific cell signaling (stress) pathways such as the antioxidant/ electrophile response element (ARE)-dependent pathways.
The KeratinoSensTM test (an ARE-Nrf2 luciferase reporter assay) is proposed to address this second key event. Skin sensitizers have been reported to induce genes that are regulated by the antioxidant response element (ARE). Small electrophilic substances such as skin sensitisers can act on the sensor protein Keap1 (Kelch-like ECH-associated protein 1), by e.g. covalent modification of its cysteine residue, resulting in its dissociation from the transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2). The dissociated Nrf2 can then activate ARE-dependent genes such as those coding for phase II detoxifying enzymes. In the KeratinoSensTM cell line activation of the Nrf2 pathway results in luciferase expression which can be quantified easily.
Positive control results:
The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at
least one concentration. The EC1.5 of the positive control was within two standard deviations of the historical mean (77 μM, 14 μM, 66 μM and 91 μM in experiment 1, 2, 3 and 4, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.39-fold, 6.87-fold, 2.16-fold and 2.38-fold in experiment 1, 2, 3 and 4, respectively).
Key result
Parameter:
other: maximal fold induction of luminescence activity (Imax)
Run / experiment:
Experiment 1
Value:
1.55
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Parameter:
other: EC 1.5 [µg/mL]
Run / experiment:
Experiment 1
Value:
5.6
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Parameter:
other: maximal fold induction of luminescence activity (Imax)
Run / experiment:
Experiment 2
Value:
1.27
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Parameter:
other: EC 1.5 [µg/mL]
Run / experiment:
Experiment 2
Remarks on result:
not determinable
Remarks:
The Imax was 1.27 and therefore no EC1.5 could be calculated.
Key result
Parameter:
other: maximal fold induction of luminescence activity (Imax)
Run / experiment:
Experiment 3
Value:
1.46
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Parameter:
other: EC 1.5 [µg/mL]
Run / experiment:
Experiment 3
Remarks on result:
not determinable
Remarks:
The Imax was 1.46 and therefore no EC1.5 could be calculated.
Key result
Parameter:
other: maximal fold induction of luminescence activity (Imax)
Run / experiment:
Experiment 4
Value:
2.11
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Parameter:
other: EC 1.5 [µg/mL]
Run / experiment:
Experiment 4
Value:
47
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
All tests passed the acceptance criteria:
- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was within two standard deviations of the historical mean (77 μM, 14 μM, 66 μM and 91 μM in experiment 1, 2, 3 and 4, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.39-fold, 6.87-fold, 2.16-fold and 2.38-fold in experiment 1, 2, 3 and 4, respectively).
- Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (8.8%, 17.3%, 6.5%, and 7.7% in experiment 1, 2, 3 and 4, respectively).

RESULTS

The test substance was evaluated for the ability to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway.

Four independent experiments were performed. The cells were in these experiments incubated with the test item in the following concentration range:

First experiment: 0.20 – 400 μg/mL (2-fold dilution series) for 48 hours ± 1 h.

Second experiment: 0.29 – 25 μg/mL (1.5-fold dilution series) for 48 hours ± 1 h.

Third experiment: 0.098 – 200 μg/mL (2-fold dilution series) for 48 hours ± 1 h.

Fourth experiment: 0.098 – 200 μg/mL (2-fold dilution series) for 48 hours ± 1 h.

The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay.

 

Experiment 1

- Precipitation was observed at the start of the incubation period at concentrations of 100 μg/mL and upwards and no precipitation was observed at the end of the incubation period.

- The test item showed toxicity. The calculated IC30 was 7.8 μg/mL and the calculated IC50 was 9.2 μg/mL.

- A dose related luminescence activity induction was observed after treatment with the test item. The Imax was 1.55 and the EC1.5 5.6 μg/mL.

- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.39 and the EC1.5 77 μM.

Experiment 2

- No precipitation was observed at the start and end of the incubation period in the 96-well plates.

- The test item showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated.

- No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imax was 1.27 and therefore no EC1.5 could be calculated.

- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 6.87 and the EC1.5 14 μM.

Experiment 3

- No precipitation was observed at the start and end of the incubation period in the 96-well plates.

- The test item showed toxicity. The calculated IC30 was 30 μg/mL and the calculated IC50 was 36 μg/mL.

- No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imax was 1.46 and therefore no EC1.5 could be calculated. The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.16 and the EC1.5 66 μM.

Experiment 4

- No precipitation was observed at the start and end of the incubation period in the 96-well plates.

- The test item showed toxicity. The calculated IC30 was 65 μg/mL and the calculated IC50 was 75 μg/mL.

- A dose related luminescence activity induction was observed after treatment with the test item. The Imax was 2.11 and the EC1.5 47 μg/mL. The induction above 1.5-fold observed at the lowest concentration was not considered biologically relevant due to the lack of dose-response.

- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.38 and the EC1.5 91 μM.

All tests passed the acceptance criteria.

Table 1: Overview Luminescence Induction and Cell Viability of the test item in Experiment 1, 2, 3 and 4

Concentration

(μg/mL)

0.2

0.39

0.78

1.6

3.1

6.3

13

25

50

100

200

400

Exp 1 luminescence

1.41

1.45

1.38

1.32

1.29

1.55***

0.01

0

0

0

0

0

Exp 1 viability (%)

102.9

113.2

106.3

96.9

101.2

94

0.1

0.2

0.3

6.8

0

0

Concentration

(μg/mL)

0.29

0.43

0.65

0.98

1.5

2.2

3.3

4.9

7.4

11

17

25

Exp 2 luminescence

0.94

0.96

0.97

1.07

1.02

0.98

1.01

1.02

1.03

0.99

1.08

1.27

Exp 2 viability (%)

131

131.5

134.5

116.7

113.7

108.5

106.1

108

110.2

109.1

103.2

105.8

Concentration

(μg/mL)

0.098

0.20

0.39

0.78

1.6

3.1

6.3

13

25

50

100

200

Exp 3 luminescence

0.99

1.01

0.99

1.06

1.08

1.06

1.11

1.19

1.46

0.04

0

0

Exp 3 viability (%)

90.8

86.5

83.7

83.8

87.5

82.9

86.7

87.1

87.2

-0.1

0

-0.1

Concentration

(μg/mL)

0.098

0.2

0.39

0.78

1.6

3.1

6.3

13

25

50

100

200

Exp 4 luminescence

2.11

1.15

1.16

1.24

1.19

1.15

1

1.32

1.26

1.53***

0.05

0

Exp 4 viability (%)

105.8

107.7

101.8

102.2

96.3

95.8

97.8

95.4

96.6

99.4

-0.4

-0.5

*** p<0.001 Student’s t test

 

Table 2: Overview Luminescence Induction and Cell Viability Positive Control EDMG in Experiment 1, 2, 3 and 4

Concentration (μM)

7.8

16

31

63

125

250

Exp 1 luminescence

1.13

1.15

1.3

1.45

1.68*

2.39***

Exp 1 viability (%)

112.9

108.9

104.9

100.1

97.3

80.6

Exp 2

luminescence

1.3

1.54**

1.57**

1.92***

2.97**

6.87**

Exp 2 viability (%)

77.8

73.1

75.3

73.9

70

55.1

Exp 3 luminescence

1.04

1

1.23

1.49

1.71***

2.16***

Exp 3 viability (%)

90.2

96

87.7

80.1

88.6

82.8

Exp 4 luminescence

1.07

1.15

1.21

1.36

1.67***

2.38***

Exp 4 viability (%)

102.8

105.4

108.7

110.8

116.9

125.4

** p<0.01 Student’s t test, *** p<0.001 Student’s t test

 

Table 3: Overview EC1.5, Imax, IC30 and IC50 values

 

EC1.5 (μM)

Imax

IC30 (μM)

IC50 (μM)

Test item Experiment 1

5.6

1.55

7.8

9.2

Test item Experiment 2

NA

1.27

NA

NA

Test item Experiment 3

NA

1.46

30

36

Test item Experiment 4

47

2.11

65

75

Pos Control Experiment 1

77

2.39

NA

NA

Pos Control Experiment 2

14

6.87

NA

NA

Pos Control Experiment 3

66

2.16

NA

NA

Pos Control Experiment 4

91

2.38

NA

NA

NA = Not applicable

 

Interpretation of results:
other: positive, to be used as part of WoE-conclusion
Conclusions:
Teh test item is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed in 2 out of 4 experiments at test concentrations < 200 μg/mL with a cell viability of >70% compared to the vehicle control.
Executive summary:

The objective of this study was to evaluate the ability of the test item to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay. The test item was dissolved in dimethyl sulfoxide at 40 mg/mL. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in the following test concentrations:

First experiment: 0.20 – 400 μg/mL (2-fold dilution series).

Second experiment: 0.29 – 25 μg/mL (1.5-fold dilution series).

Third experiment: 0.098 – 200 μg/mL (2-fold dilution series).

Fourth experiment: 0.098 – 200 μg/mL (2-fold dilution series).

The highest test concentration was the highest dose required in the current guideline. No precipitate was observed at any dose level tested. Four independent experiments were performed. All experiments passed the acceptance criteria.

In the first experiment, the test item showed toxicity (IC30 value of 7.8 μg/mL and IC50 value of 9.2 μg/mL). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 value of 5.6 μg/mL) was measured. The maximum luciferase activity induction (Imax) was 1.55-fold. This led to an individual run conclusion of positive, since induction above 1.5-fold was observed at test concentration with cell viability >70%. In the second experiment, the test item showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured. The maximum luciferase activity induction (Imax) was 1.27-fold. The led to an individual run conclusion of inconclusive since negative results were obtained with concentration <200 μg/mL and no cytotoxicity (< 70% viability) was observed. As the first and second experiment were not concordant an additional third experiment was conducted. In the third experiment the test item showed toxicity (IC30 value of 30 μM and IC50 value of 36 μM). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured. The maximum luciferase activity induction (Imax) was 1.46-fold leading to an individual run conclusion of negative. Since no final conclusion could be made based on these three results, an additional fourth experiment was conducted. In the fourth experiment, the test item showed toxicity (IC30 value of 65 μg/mL and IC50 value of 75 μg/mL). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 value of 47 μg/mL) was measured. The maximum luciferase activity induction (Imax) was 2.11-fold. This led to an individual run conclusion of positive, since induction above 1.5-fold was observed at test concentration with cell viability >70%. The test item is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed in 2 out of 4 experiments at test concentrations < 200 μg/mL with a cell viability of >70% compared to the vehicle control. In conclusion, the test substance is classified as positive (biologically relevant activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

OECD 442E – Annex II ‘In Vitro Skin Sensitisation: U937 Cell Line Activation Test (USENS ™)

The objective of this study is to evaluate the ability of the test item to increase the expression levels of CD86 cell surface marker in the U937 cell line in the U937 cell line activation Test (U-Sens™) assay. The test item was dissolved in dimethyl sulfoxide. Three independent experiments were performed. In the first experiment the test item showed toxicity (CV70 value of 78 μg/mL) and a biologically relevant induction of the CD86 activity (EC150 value of 22 μg/mL) was measured. The individual run conclusion is considered No Conclusion, since the S.I. of CD86 is higher or equal to 150% at the highest non-cytotoxic concentration only. In the second experiment the test item showed toxicity (CV70 value of 86 μg/mL) and a biologically relevant, induction of the CD86 activity (EC150 value of 63 μg/mL) was measured. The individual run conclusion is considered Positive. An additional third experiment was performed, since the first two experiments were not concordant. In the third experiment the test item showed toxicity (CV70 value of 61 μg/mL) and a biologically relevant, induction of the CD86 activity (EC150 value of < 20 μg/mL) was measured. The individual run conclusion is considered Positive. The test item is classified as Positive in the U-Sens™ assay since positive results (> 150% increase) were observed at test concentrations with a cell viability of >70% compared to the vehicle control. In conclusion, the test item is classified as positive (increase in the expression levels of CD86 cell surface marker in the U937 cell line) under the experimental conditions described.

 

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

The objective of this study was to evaluate the ability of the test item to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay. The test item was dissolved in dimethyl sulfoxide.

The highest test concentration was the highest dose required in the current guideline. Four independent experiments were performed. All experiments passed the acceptance criteria.

In the first experiment, the test item showed toxicity (IC30 value of 7.8 μg/mL and IC50 value of 9.2 μg/mL). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 value of 5.6 μg/mL) was measured. The maximum luciferase activity induction (Imax) was 1.55-fold. This led to an individual run conclusion of positive, since induction above 1.5-fold was observed at test concentration with cell viability >70%. In the second experiment, the test item showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured. The maximum luciferase activity induction (Imax) was 1.27-fold. The led to an individual run conclusion of inconclusive since negative results were obtained with concentration <200 μg/mL and no cytotoxicity (< 70% viability) was observed. As the first and second experiment were not concordant an additional third experiment was conducted. In the third experiment the test item showed toxicity (IC30 value of 30 μM and IC50 value of 36 μM). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured. The maximum luciferase activity induction (Imax) was 1.46-fold leading to an individual run conclusion of negative. Since no final conclusion could be made based on these three results, an additional fourth experiment was conducted. In the fourth experiment, the test item showed toxicity (IC30 value of 65 μg/mL and IC50 value of 75 μg/mL). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 value of 47 μg/mL) was measured. The maximum luciferase activity induction (Imax) was 2.11-fold. This led to an individual run conclusion of positive, since induction above 1.5-fold was observed at test concentration with cell viability >70%. The test substance is classified as positive (biologically relevant activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

As an overall conclusion, the substance has to be classified as skin sensitising based on two positive results in the in vitro test battery.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. Based on this data, the substanceis considered to be classified for skin sentisation (Cat. 1) under Regulation (EC) No 1272/2008, as amended for the twelth time in Regulation (EC) No 2019/521.