Registration Dossier

Administrative data

Description of key information

In an in chemico direct peptide reactivity assay (DPRA) according to OECD Guideline 442 C (reference 7.4.1-1), the test item showed moderate reactivity towards cysteine or lysine, respectively under the given conditions. In an in vitro skin sensitisation assay (ARE-Nrf2 Luciferase Test Method, KeratinoSens) according to OECD Guideline TG 442 D (reference 7.4.1 -2), the test item did not induce luciferase activity in at least two independent experimental runs. In an in vitro assay according to OECD TG 442E (h-CLAT) addressing the third key event of skin sensitisation, no activation of dendritic cells was observed.

Based on the results from the in chemico and in vitro studies and considering the AOP “2 out of 3” the test item in predicted as non-skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2018-05-14 to 2018-05-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154
Version / remarks:
2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
direct peptide binding assay
Details on study design:
Skin sensitisation (In chemico test system) - Details on study design:

Preparation of the Test Item:
The test item was freshly prepared immediately prior to use, unless stability data demonstrate the acceptability of storage. The test item was pre-weighed into a glass vial and was dissolved in an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared. A factor of 1.159 was used to correct for the purity of the test item.

Controls:
Reference controls, co-elution controls and a positive control (PC) were set up in parallel to the test item in order to confirm the validity of the test.

Positive Control:
Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.

Co-elution Control:
Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides.

Reference Control
Reference controls (RCs) were set up in parallel to sample preparation in order to verify the validity of the test run. Reference control A was prepared using acetonitrile in order to verify the accuracy of the calibration
curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run
Reference control B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each HPLC run.
Reference control C was set up for the test item and the positive control. RC C for the positive control was prepared using acetonitrile. RC C for the test item was prepared using the respective solvent used to solubilise the test item. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples.

Peptides:
19.55 mg cysteine peptide (JPT Peptide Technologies GmbH; > 95%) with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (37.19 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
18.08 mg lysine peptide (JPT Peptide Technologies GmbH; > 95%) with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (34.34 mL) to reach a concentration of 0.667 mM.
All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.

Dose Groups:
Reference Control C (solvent control)
Test Item :100 mM stock solution
Positive Control: 100 mM stock solution

Pre-Experiments:
Solubility of the test item was determined prior to the main experiment and was tested at the highest final concentration applied in the study (100 mM). Solubility was investigated in the following solvents suitable for the test:
- acetonitrile
- dist. water
- dist. water : acetonitrile 1:1 (v/v)
The test item was not soluble in either acetonitrile or dist. water. The test item was completely soluble in dist. water : acetonitrile 1:1 (v/v) , therefore, dist. water : acetonitrile 1:1 (v/v) was chosen as suitable vehicle for the main experiments.

Experimental Procedure
The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. Reference controls, co-elution controls as well as the positive control were set up in parallel. Test item solutions were inspected on a visual basis for the formation of precipitates, turbidity and phase separation prior and after HPLC analysis.
After the incubation period of 24 ± 2 h the test item was analysed in triplicate for both peptides using HPLC.

Preparation of the HPLC Standard Calibration Curve:
A standard calibration curve was generated for both, the cysteine and the lysine peptide. Peptide standards were prepared in a solution of 20% acetonitrile : 80% buffer ( v / v ) using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide (dilution buffer (DB)). A serial dilution of the peptide stock solution (0.667 mM) using the respective DB was performed, resulting in 7 calibration solutions.

HPLC Preparation and Analysis:
Peptide depletion was monitored by HPLC coupled with an UV detector at λ = 220 nm using a reversed-phase HPLC column (Zorbax SB-C-18 2.1 mm x 100 mm x 3.5 micron) as preferred column. The entire system was equilibrated at 30 °C with 50% phase A and 50% phase B for at least 2 hours before running the analysis sequence. The HPLC analysis was performed using a flow rate of 0.35 mL/min and a linear gradient from 10% to 25% acetonitrile over 10 minutes, followed by a rapid increase to 90% acetonitrile. The column was re-equilibrated under initial conditions for 7 minutes between injections. Equal volumes of each standard, sample and control were injected. HPLC analysis for the cysteine and lysine peptide was performed concurrently (if two HPLC systems were available) or on separate days. If analysis was conducted on separate days all test chemical solutions were freshly prepared for both assays on each day. The analysis was timed to assure that the injection of the first sample started 22 to 26 hours after the test chemical was mixed with the peptide solution. The HPLC run sequence was set up in order to keep the HPLC analysis time less than 30 hours.

Data Analysis:
The concentration of the cysteine and lysine peptide was determined in each sample from absorbance at λ = 220 nm, measuring the area of the appropriated peaks (peak area (PA)) and calculating the concentration of peptide using the linear calibration curves derived from the standard solutions. The percent peptide depletion (PPD) was calculated. The absorbance at λ = 258 nm was also monitored for the samples of the test item and the reference controls as a co-elution control. The ratio of the peak areas (220 nm / 258 nm) was
checked for consistency between reference control and test item samples. If this ratio was not consistent a co-elution was assumed and the evaluation would be adjusted accordingly. Sensitising potential of the test item is predicted from the mean cysteine and lysine PPD value.

Prediction Model:
The test item is considered positive, i.e. to be a skin sensitiser, if the mean depletion of both peptides exceeds the threshold of the respective prediction model. Negative depletion is considered as “0” when calculating the mean. Sensitizing potential might not be predictable if the test item was incubated using a concentration differently from 100 mM. By using the prediction model 1 (cysteine 1:10 / lysine 1:50 prediction model), see tabe 3) the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers. Application of the prediction model for assigning a test item to a reactivity class (i.e. low, moderate or high reactivity) may perhaps prove useful to inform potency assessment within the framework of an IATA. In the framework of an IATA the test substance may be considered as non-sensitiser to skin, if the mean depletion of both peptides is below 6.38%. In case of co-elution of the test item with a peptide peak, the peak cannot be integrated correctly and the calculation of the PPD is not possible. If severe co-elution occurs with both peptides then the analysis was reported as "inconclusive". In cases where the co-elution occurs only with the lysine peptide prediction model 2 can be applied (cysteine 1:10 prediction model). A single HPLC analysis for both the cysteine and the lysine peptide should be sufficient for a test
chemical when the result is unequivocal. However, in cases of results close to the threshold used to discriminate between positive and negative results (i.e. borderline results), additional testing may be necessary. In situations where the mean percent depletion falls in the range of 3% to 10% for the cysteine 1:10/lysine 1:50 prediction model or the cysteine percent depletion falls in the range of 9% to 17% for the cysteine 1:10 prediction model, a second run should be considered, as well as a third one in case of discordant results between the first two runs.

Acceptance Criteria:
The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.
The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.
Positive control results:
The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 65.88%.
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
cysteine run
Value:
3.96
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
lysine run
Value:
77.88
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: mean peptide depleation of both peptides [%]
Run / experiment:
1st experiment
Value:
40.92
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Pre-Experiments:
Solubility of the test item was determined prior to the main experiment. The test item was soluble in dist. water : acetonitrile 1:1 (v/v). No turbidity, precipitation and phase separation was observed for the test item solution. All test item preparations of the main experiment were prepared using dist. water : acetonitrile 1:1 (v/v). All test item solutions were freshly prepared immediately prior to use.

Co-elution with the Peptide Peaks:
No co-elution of the test item with any of the peptide peaks was observed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 1: Results

Cysteine and Lysine Values of the Calibration Curve

Sample

Cystein Peptide

Lysine Peptide

Peak Area at 220 nm

Peptide Concentration [mM]

Peak Area at 220 nm

Peptide Concentration [mM]

STD1

15.8430

0.5340

16.1480

0.5340

STD2

8.0650

0.2670

8.0680

0.2670

STD3

4.0830

0.1335

4.0960

0.1335

STD4

2.0190

0.0667

2.0510

0.0667

STD5

0.9700

0.0334

1.0420

0.0334

STD6

0.4700

0.0167

0.5180

0.0167

STD7

0.0000

0.0000

0.0000

0.0000

Table 2: Depletion of the Cysteine Peptide

Cysteine Peptide

Sample

Peak Area at 220 nm

Peptide Concentration [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

4.4450

0.1486

70.62

70.09

0.62

0.88

4.5010

0.1505

70.25

4.6270

0.1547

69.42

Test Item

15.5030

0.5203

2.73

3.96

1.09

27.42

15.2440

0.5116

4.35

15.1750

0.5093

4.79

Table 3: Depletion of the Lysine Peptide

Lysine Peptide

Sample

Peak Area
at 220 nm

Peptide Conc. [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

5.6780

0.1872

62.87

61.66

1.15

1.86

5.8860

0.1941

61.51

6.0270

0.1988

60.59

Test Item

3.4580

0.1137

77.20

77.88

0.64

0.82

3.3400

0.1098

77.98

3.2660

0.1073

78.46

Table 4: Categorization of the Test Item

Prediction Model

Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Item Ratio: 1:10 and 1:50)

Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)

Test Substance

Mean Peptide Depletion [%]

Reactivity Category

Prediction

Mean Peptide Depletion [%]

Reactivity Category

Prediction

Test Item

40.92

Moderate Reactivity

sensitiser

--*

--*

--*

Positive Control

65.88

High Reactivity

sensitiser

70.09

Moderate Reactivity

sensitiser

* = not applicable

Interpretation of results:
other: peptide depletion
Conclusions:
In this study under the given conditions the test item showed moderate reactivity towards both peptides. The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation Adverse Outcome Pathway (AOP).
Executive summary:

A study according OECD TG 442 C was conducted for detection of the sensitising potential of the test item by quantifying its reactivity towards synthetic peptides containing either lysine or cysteine.

The test item was dissolved in dist. water : acetonitrile 1:1 (v/v), based on the results of the pre-experiments. Considering the molecular weight of 872.45 g/mol of the test item, a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently, samples were analysed by HPLC. All test item solutions were freshly prepared immediately prior to use.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (excluding the co-elution control). Samples were centrifuged prior to the HPLC analysis. Since it cannot be determined if the precipitate resulted from the test item or the peptide, the given peak areas and corresponding peptide values can only be considered as an estimation of the peptide depletion.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as not relevant.

No co-elution of test item with the cysteine or lysine peptide peak was observed. A possible sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C dist. Water : Acetonitrile 1:1 (v/v)).

The 100 mM stock solution of the test item showed moderate reactivity towards the synthetic peptides. The mean depletion of both peptides was 40.92% and, thus, exceeded the threshold of 6.38%. Even though a precipitate was observed in the cysteine experiment a positive result can still be used. Based on the cysteine 1:10 / lysine 1:50 prediction model the test item can be considered as a skin sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 65.88%.

In this study under the given conditions the test item showed moderate reactivity towards both peptides. The test item is considered as skin sensitiser in this in vitro assay. However, the data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation Adverse Outcome Pathway (AOP).

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2018-06-13 to 2018-06-25
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:
2015
Deviations:
no
Qualifier:
according to
Guideline:
other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
activation of keratinocytes
Details on study design:
Skin sensitisation (In vitro test system) - Details on study design:
Cell line:
The test was carried out using the transgenic cell line KeratinoSens™, a cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and were used for routine testing. Only cells at a low passage number <25 (P 11 in experiment 1; P 02 in experiment 2) were used. Cells were cultured in 75 cm2 culture flasks (Greiner) in maintenance medium at 37 ± 1°C and 5% CO2 in a humidified incubator. For test item exposure, cells were cultured in medium for test item exposure.

Test Item Exposure Medium:
Dulbecco’s Modified Eagle Medium (GlutaMAX™) with 1.0 g/L D-glucose and 1 mM Na-Pyruvate. The medium was supplemented with the following components: 1% fetal bovine calf serum

Preparation of the Test Item:
All test item solutions were freshly prepared immediately prior to use. The test item was dissolved in dimethyl sulfoxide. A stock solution of 200 mM was prepared by pre-weighing the test material into a glass vial. A factor of 1.159 to correct for the purity of the test item was used. Vortex mixing was used to aid solubilisation. Based on the stock solution a set of twelve master solutions in 100% solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a 4% share of the solvent. These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.

Dose Groups:
1. Negative Control: 1% (v/v) DMSO in test item exposure medium
2. Positive Control: CA: 4 μM, 8 μM, 16 μM; 32 μM; 64 μM
3. Test Item: 12 concentrations of the test item
Each concentration step of the test item and the positive control was assessed in three replicates in every independent run. The negative control was assessed using six replicates per 96-well plate in every independent run.

Blank:
A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.

Negative Control:
DMSO at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate. The preparation of the negative control was carried out analogous to the test item.

Positive Control:
Cinnamic aldehyde was used as positive control. CA was dissolved in DMSO at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 μM – 64 μM. The final concentration of the solvent DMSO was 1% (v/v) for all wells.

Experimental Procedure:
A cell suspension of 8 × 10E4 cells/mL in assay medium was prepared. 125 μL of the cell suspension corresponding to 1 × 10E4 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel, cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability. After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 μL test item exposure medium. 50 μL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item. All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.

Luciferase activity:
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently, 20 μL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light. Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 μL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1 sec. before assessing the luciferase activity for 2 sec. This procedure was repeated for each individual well.

Cell viability:
For the cell viability plate the medium was replaced with 200 μL test item exposure medium. 27 μL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 200 μL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5% CO2 over the weekend (experiment 1 and 2). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.

Prediction Model:
A KeratinoSens™ prediction is considered positive if the following conditions will be met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is <1000 μM
- an apparent overall dose-response for luciferase induction

Acceptance Criteria:
The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 μM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition.
Positive control results:
The luciferase activity induced by the positive control at a concentration of 64 µM was 3.21 (experiment 1) and 5.22 (experiment 2).
Key result
Parameter:
other: luciferase activity
Run / experiment:
1st experiment
Value:
1.32
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 7.81 µM
Key result
Parameter:
other: luciferase activity
Run / experiment:
2nd experiment
Value:
2.44
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 7.81 µM
Key result
Parameter:
other: EC1.5 [µM]
Run / experiment:
2nd experiment
Value:
5.06
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 1: Results of the Cytotoxicity Measurement

 

Concentration [µM]

Cell Viability [%]

Experiment 1

Experiment 2

Mean

SD

Solvent Control

-

100

100

100

0.0

Positive Control

4.00

97.8

107.0

102.4

6.5

8.00

96.6

111.9

104.3

10.8

16.00

97.1

114.0

105.5

12.0

32.00

97.6

116.6

107.1

13.4

64.00

88.9

112.8

100.9

16.9

Test Item

0.98

91.6

105.9

98.7

10.1

1.95

91.0

98.0

94.5

4.9

3.91

86.8

101.1

94.0

10.2

7.81

92.3

59.1

75.7

23.4

15.63

25.7

2.3

14.0

16.6

31.25

1.5

0.0

0.7

1.0

62.50

0.4

0.6

0.5

0.1

125.00

0.3

0.2

0.2

0.1

250.00

0.8

0.3

0.6

0.4

500.00

1.7

0.2

0.9

1.1

1000.00

2.6

0.3

1.5

1.6

2000.00

2.3

0.3

1.3

1.4

Table 2: Induction of Luciferase Activity Experiment 1

Experiment 1

Concentration [µM]

Fold Induction

Significance

Rep. 1

Rep. 2

Rep. 3

Mean

SD

Solvent Control

-

1.00

1.00

1.00

1.00

0.00

 

Positive Control

4.00

1.12

1.12

1.24

1.16

0.07

 

8.00

1.21

1.26

1.10

1.19

0.08

 

16.00

1.46

1.46

1.38

1.43

0.05

 

32.00

1.93

1.91

1.71

1.85

0.12

*

64.00

2.75

3.08

3.80

3.21

0.54

*

Test Item

0.98

0.89

0.94

0.92

0.92

0.02

 

1.95

0.91

0.91

0.91

0.91

0.00

 

3.91

0.96

0.89

0.98

0.94

0.05

 

7.81

1.24

1.25

1.46

1.32

0.12

 

15.63

1.02

1.15

1.46

1.21

0.23

 

31.25

0.02

0.02

0.02

0.02

0.00

 

62.50

0.00

0.00

0.00

0.00

0.00

 

125.00

0.00

0.00

0.00

0.00

0.00

 

250.00

0.00

0.00

0.00

0.00

0.00

 

500.00

0.00

0.00

0.00

0.00

0.00

 

1000.00

0.00

0.00

0.00

0.00

0.00

 

2000.00

0.00

0.00

0.00

0.00

0.00

 

* = significant induction according to Student’s t-test, p < 0.05

Table 3 Induction of Luciferase Activity Experiment 2

Experiment 2

Concentration [µM]

Fold Induction

Significance

Rep. 1

Rep. 2

Rep. 3

Mean

SD

Solvent Control

-

1.00

1.00

1.00

1.00

0.00

 

Positive Control

4.00

1.25

1.27

1.23

1.25

0.02

 

8.00

1.28

1.25

1.58

1.37

0.18

 

16.00

1.33

1.40

1.47

1.40

0.07

 

32.00

2.02

2.03

2.47

2.17

0.25

*

64.00

4.42

5.04

6.19

5.22

0.90

*

Test Item

0.98

0.85

0.91

0.78

0.85

0.06

 

1.95

1.04

0.73

0.85

0.87

0.16

 

3.91

1.28

1.07

0.97

1.11

0.16

 

7.81

2.47

2.34

2.51

2.44

0.09

*

15.63

0.48

0.11

0.17

0.25

0.20

 

31.25

0.00

0.00

0.00

0.00

0.00

 

62.50

0.00

0.00

0.00

0.00

0.00

 

125.00

0.00

0.00

0.00

0.00

0.00

 

250.00

0.00

0.00

0.00

0.00

0.00

 

500.00

0.00

0.00

0.00

0.00

0.00

 

1000.00

0.00

0.00

0.00

0.00

0.00

 

2000.00

0.00

0.00

0.00

0.00

0.00

 

* = significant induction according to Student’s t-test, p < 0.05

Table 4 Induction of Luciferase Activity – Overall Induction

 

Concentration [µM]

Fold Induction

Significance

Experiment 1

Experiment 2

Mean

SD

Solvent Control

-

1.00

1.00

1.00

0.00

 

Positive Control

4.00

1.16

1.25

1.20

0.06

 

8.00

1.19

1.37

1.28

0.13

 

16.00

1.43

1.40

1.42

0.02

 

32.00

1.85

2.17

2.01

0.23

*

64.00

3.21

5.22

4.21

1.42

 

Test Item

0.98

0.92

0.85

0.88

0.05

 

1.95

0.91

0.87

0.89

0.03

 

3.91

0.94

1.11

1.03

0.11

 

7.81

1.32

2.44

1.88

0.79

 

15.63

1.21

0.25

0.73

0.67

 

31.25

0.02

0.00

0.01

0.01

 

62.50

0.00

0.00

0.00

0.00

 

125.00

0.00

0.00

0.00

0.00

 

250.00

0.00

0.00

0.00

0.00

 

500.00

0.00

0.00

0.00

0.00

 

1000.00

0.00

0.00

0.00

0.00

 

2000.00

0.00

0.00

0.00

0.00

 

* = significant induction according to Student’s t-test, p<0.05

Table 5 Additional Parameters

Parameter

Experiment 1

Experiment 2

Mean

SD

EC1.5[µM]

n.a.

5.06

n.a.

n.a.

Imax

1.32

2.44

1.88

0.79

IC30[µM]

10.43

6.80

8.62

2.56

IC50[µM]

12.78

9.07

10.92

2.62

n.a.: not applicable

Table 6 Acceptance Criteria

Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

CV Solvent Control

< 20%

8.9

pass

15.1

pass

No. of positive control concentration steps with significant luciferase activity induction >1.5

≥ 1

2.0

pass

2.0

pass

EC1.5 PC

7 < x < 34 µM

18.59

pass

18.06

pass

Induction PC at 64 µM

2.00 < x < 8.00

3.21

pass

5.22

pass

 

Table 7 Historical Data

Acceptance Criterion

Range

Mean

SD

N

CV Solvent Control

< 20%

11.6

3.5

96

No. of positive control concentration steps with significant luciferase activity induction >1.5

≥ 1

2.4

0.6

96

EC1.5 PC

7 < x < 34 µM

18.5

6.0

96

Induction PC at 64 µM

2.00 < x < 8.00

3.8

1.5

96

Interpretation of results:
other: no activation of keratinocytes
Conclusions:
In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser. The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.
Executive summary:

A study according OECD TG 442 D was conducted which enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

In the present study the test item was dissolved in DMSO. Based on a molecular weight of 872.45 g/mol a stock solution of 200 mM was prepared. Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:

2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 μM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement. In the first experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. In the second experiment, a max luciferase activity (Imax) induction of 2.44 was determined at a test item concentration of 7.81 μM. The corresponding cell viability was 59.1%. No further significant luciferase induction > 1.5 was found in the tested concentration range. The calculated EC1.5 was <1000 μM (5.06 μM). No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Under the condition of this study the test item is therefore considered as non sensitiser.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2018-08-23 to 2018-10-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals, No. 442E: In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation”, adopted 25 June 2018
Version / remarks:
2018
Deviations:
no
Qualifier:
according to
Guideline:
other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
activation of dendritic cells
Details on study design:
Skin sensitisation (In vitro test system) - Details on study design:

Preparation of the Test Item:
The test item was freshly prepared immediately prior to use. The test item was soluble in dimethyl sulfoxide (DMSO) at a concentration of 500 mg/mL. Vortex mixing and sonication were used to aid solubilisation. Stock solutions were prepared by diluting the highest soluble concentration seven times with a constant dilution factor of 1:2. The working stock solutions were prepared by diluting each stock solution 250 times with cell culture medium. No precipitation, turbidity or phase separation was observed when diluted 1:250 in cell culture medium. The working stock solutions were applied to the cells by adding equal volumes of each solution to prepared cells, resulting in a further 1:2 dilution of the working solutions. The solvent was present at a constant volume ratio of 0.2% (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.

Controls:
A medium control, a solvent control, and a positive control were set up in parallel in order to confirm the validity of the test.

Medium Control:
A medium control was included in the test.

Solvent Controls:
Solvent controls were included in the test. The solvent controls were set up depending on the appropriate solvent previously determined. Since the test item was solubilized in DMSO, a DMSO control served as solvent control for the test item. Since the positive control was solubilized in DMSO, a DMSO control was included and served as solvent control for the positive control. The solvent controls were diluted according to the procedure described for the test item, resulting in a final concentration of 0.2% (v/v) for DMSO.

Positive Control:
2,4-dinitrochlorobenzene (DNCB) at a final concentration of 4 μg/mL (alternatively at the concentration of the CV75) was tested concurrently with the test item. DNCB was dissolved in DMSO and diluted according to the procedure described for the test item , resulting in a final DMSO concentration of 0.2% (v/v).

FACS:
FACS: BD Canto II
Software: BD FACS DIVA 6.0
Voltage Settings: FSC: 300 V
SSC: 250 V FITC: 400 V PI: 420 V
Threshold value of FSC: 5000
Compensation: PI - 14 % FITC
FITC – 0 % PI

Cell line:
The test was carried out using THP-1 cells (ATCC® TIB-202TM), an acute human monocytic leukemic cell line used as a surrogate for DC. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and subcultured at least 2 weeks before they were used in the in vitro h-CLAT test. Cells at passage number (<30) were used. Cells are routinely passaged every 2-3 days at a density of 0.1 – 0.2 x 10E6 cells/mL.
Cells were cultured in 75 cm2 culture flasks (Greiner) in Roswell Park Memorial Institute medium supplemented with 10% fetal bovine serum, 25 mM HEPES, L-glutamine, 0.05 mM 2-mercaptoethanol and 100 U/ml penicillin/ 100 μg/mL streptomycin at 37 ± 1°C and 5% CO2.

Dose Groups:
1. Medium Control: cell culture medium
2. Solvent Control: 0.2% DMSO (v/v) in cell culture medium
3. Positive Control: 4 μg/mL DNCB
4. Test Item: 8 concentrations of the test item (dose finding assay/ main experiment) dose finding assay 1 and 2:
1000, 500, 250, 125, 62.50, 31.25, 15.63, 7.81 μg/mL
main experiment 1 and 2:
710.99, 592.49, 493.74, 411.45, 342.88, 285.73, 238.11, 198.42 μg/mL

Pre-Experiments:
Reactivity Check of the Cells Stock
Prior to testing, the quality of freshly thawed cell batch was checked by monitoring the doubling time and checking the reactivity towards positive controls. For the reactivity check of the cell batch additional negative and positive controls were included. DNCB at a final concentration of 4 μg/mL and nickel sulphate at a final concentration of 100 μg/mL served as positive control while lactic acid at a final concentration of 1000 μg/mL served as negative control. Cells were accepted when both, DNCB and nickel sulphate produce a positive response for CD86 and CD54, and lactic acid produces a negative response for CD86 and CD54.
Solvent Finding
Solubility of the test item was determined prior to the main experiment. The test item was dissolved in 0.9% NaCl at a final concentration of 500 mg/mL. Test items not soluble in 0.9% NaCl solution were dissolved in DMSO at a concentration of 500 mg/mL. If the test item was not soluble in DMSO, other solvents (e.g. THF) were used. It was taken care that the test chemical is dissolved or stably dispersed in the chosen solvent and that it does not interfere with the test design. If the test item was not soluble in DMSO or a different organic solvent at 500 mg/mL, the highest soluble concentration was tested by diluting the solution from 500 mg/mL with a constant factor of 1:2 up to a minimal concentration of 1 mg/mL.

Experimental Procedure:
Dose Finding Assay
Starting from 500 mg/mL solutions of the test chemicals, eight stock solutions (eight concentrations) were prepared, by 2-fold serial dilutions using the corresponding solvent. These stock solutions were further diluted 250-fold into culture medium (working solutions). The working solutions were finally used for treatment by adding an equal volume of working solution to the volume of THP-1 cell suspension in a 96-well plate to achieve a further 2-fold dilution. For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of 0.1 – 0.2 x 10E6 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation and were re-suspended in fresh culture medium at a density of 2 x 10E6 cells/mL. Then, 500 μL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 10E6 cells/well). The solvent controls and the test item working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2.
After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The supernatant was discarded and the remaining cells were washed twice with Dulbecco’s phosphate buffered saline (DPBS) containing 0.1% bovine serum albumin (BSA; i.e. FACS buffer). After washing, cells were re-suspended in 600 μL FACS buffer. 200 μL of the cell suspension were transferred into a FACS tube and stained by using propidium iodide (PI) solution at a final concentration of 0.625 μg/mL.
The PI uptake of the cells and therefore cytotoxicity was analysed immediately after the staining procedure by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ > 650 nm. A total of 10,000 living (PI negative) cells were acquired and cell viability was calculated for each test concentration. The CV75 value, i.e. the concentration showing 75% cell survival, was calculated by log-linear interpolation. The CV75 value was used to calculate the concentration range of the test item for the main experiment.

CD54 and CD86 Expression:
The test item was dissolved using DMSO as determined in the pre-experiment. Based on the concentration of the pre-determined CV75 value 8 concentrations of the test item were defined for the measurement of the surface marker expression. If the CV75 could not be determined due to insufficient cytotoxicity of the test item in the dose finding assay, the highest soluble concentration of the test item prepared with each solvent was used as starting dose.
The test item was diluted to the concentration corresponding to 500-fold of the 1.2 × CV75. Then, 1.2-fold serial dilutions were made using the corresponding solvent to obtain the 8 stock solutions to be tested. The stock solutions were further diluted 250-fold into the culture medium (working solutions). These working solutions were finally used for cell treatment with a further final 2-fold dilution factor.
For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of 0.1 – 0.2 x 10E6 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation (125 x g) and were re-suspended in fresh culture medium at a density of 2 x 10E6 cells/mL. Then, 500 μL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 10E6 cells/well).
The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2.
After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The following steps were carried out on ice with pre-cooled buffers and solutions. The supernatant was discarded and the remaining cells were washed twice with FACS buffer. After washing, cells were blocked using 600 μL of a FcR blocking buffer (FACS buffer containing 0.01% (w/v) Globulin Cohn Fraction) and incubated at 4 °C for 15 min. After blocking, cells were split in three aliquots into a 96-well V-bottom plate. After centrifugation (approx. 250 x g), cells were stained with 50 μL of FITC-labelled anti-CD86, anti-CD54, or mouse IgG1 (isotype) antibodies in the dark for 30 min. All antibodies were diluted in FACS buffer at an appropriate manner. After washing with FACS buffer two times, cells were re-suspended in FACS buffer and PI solution was added. PI staining was done just prior to the measurement by adding PI solutions to each sample (final concentration of PI was 0.625 μg/mL).
The expression levels of CD86 and CD54 as well as cell viability were analysed by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ = 530 nm ± 15 nm for FITC and λ > 650 nm for PI. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 were calculated.

Prediction Model:
For CD86/CD54 expression measurement, each test item was tested in at least two independent runs to derive a single prediction. Each independent run was performed on a different day or on the same day provided that for each run: independent fresh stock solutions and working solutions of the test chemicals and antibody solutions were prepared and independently harvested cells were used.
Sensitising potential of the test item was predicted from the mean percentage expression of CD86 and CD54. Any test chemical tested by the h-CLAT was considered positive if the RFI of CD86 was equal to or greater than 150% at any tested dose at a cell viability ≥ 50% in at least two independent runs or if the RFI of CD54 was equal to or greater than 200% at any tested dose at a cell viability ≥ 50% in at least two independent runs or if the RFIs of both the CD86 and CD54 were equal to or are greater than 150% and 200% respectively at any tested dose at a cell viability ≥ 50% in at least two independent runs. In case of not concordant results a third run should be conducted to make the final prediction. Otherwise the results were considered as inconclusive.
A negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x CV75 is <90%. In contrast, a positive test outcome was accepted irrespective of cell viabilities >90% at a concentration of 1.2 x CV75. If no CV75 could be derived negative test results can be accepted when the test item is tested at the highest soluble concentration (5000 μg/mL for 0.9% NaCl solution; 1000 μg/mL for DMSO or a different organic solvent) even if the cell viability is >90%.

Acceptance criteria:
The test meets acceptance criteria if:
- the cell viability of the solvent controls is >90%,
- the cell viability of at least four tested doses of the test item in each run is >50%,
- the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
- the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
- the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%.
Positive control results:
The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (263% experiment 1; 345% experiment 2) and 200% for CD54 (225% experiment 1; 264% experiment 2) were clearly exceeded.
Key result
Parameter:
other: relative fluorescence intensity CD86 [%]
Run / experiment:
1st experiment
Value:
64
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 198.42 µg/mL
Key result
Parameter:
other: relative fluorescence intensity CD54 [%]
Run / experiment:
1st experiment
Value:
96
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 592.49 µg/mL
Key result
Parameter:
other: relative fluorescence intensity CD86 [%]
Run / experiment:
2nd experiment
Value:
88
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 342.88 µg/mL
Key result
Parameter:
other: relative fluorescence intensity CD54 [%]
Run / experiment:
2nd experiment
Value:
115
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 592.49 µg/mL
Other effects / acceptance of results:
Doubling time of the cells was monitored and found to be 39.9 h which is within the doubling time range specified by the manufacturer (35 - 50 h).

DEMONSTRATION OF TECHNICAL PROFICIENCY: The positive controls DNCB and NiSO4 led to upregulation of the cell surface markers CD54 and CD86. The negative control LA did not induce an upregulation of CD54 and CD86. The cell batch was accepted for further testing.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 1: Results of the Cell Batch Activation Test

Sample

Concentration
[µg/mL]

CD86

CD54

Activated

Pass /Fail

Cell Viability [%]

RFI

Threshold OECD TG 442E

Cell Viability [%]

RFI

Threshold OECD TG 442E

yes/no

DNCB

4 µg/mL

87.5

373

>150

88.1

358

>200

yes

pass

NiSO4

100 µg/mL

83.5

295

>150

82.1

603

>200

yes

pass

LA

1000 µg/mL

95.7

81

150

95.8

100

200

no

pass

Table 2: Results of the Dose Finding Assay

Sample

Experiment 1

Experiment 2

Concentration applied [µg/mL]

Cell Viability [%]

Concentration applied [µg/mL]

Cell Viability [%]

Medium Control

--

--

96.80

--

94.30

Solvent Control

DMSO

--

97.10

--

95.00

Test item

C8

7.81

96.60

7.81

93.70

C7

15.63

97.40

15.63

93.00

C6

31.25

96.60

31.25

92.50

C5

62.50

95.80

62.50

86.70

C4

125.00

91.40

125.00

82.80

C3

250.00

83.20

250.00

77.20

C2

500.00

81.60

500.00

78.60

C1

1000.00

56.10

1000.00

63.00

Calculated CV75 [µg/mL]

598.25

586.73

Mean CV75 [µg/mL]

592.49

SD CV 75 [µg/mL]

8.15

Table 3 CD54 and CD86 Expression Experiment 1

Sample

Conc.
[μg/mL]

Cell Viability [%]

Mean Fluorescence Intensity

corrected Mean Fluorescence Intensity

Relative Flourescence Intensity (RFI)

Ratio Isotype IgG1 to [%]

CD86

CD54

Isotype IgG1

CD86

CD54

Isotype IgG1

CD86

CD54

CD86

CD54

CD86

CD54

Medium Control

-

93.1

93.3

93.9

3072

1372

779

2293

593

90

91

394

176

Solvent Control

0.20%

93.1

93.6

93.2

3290

1383

734

2556

649

100

100

448

188

DNCB

4.00

80.7

81.7

80.5

7434

2170

709

6725

1461

263

225

1049

306

Test item

710.99*

90.9

89.1

92.1

6154

4268

1201

4953

3067

194

473

512

355

592.49

70.5

68.5

69.8

2380

1464

843

1537

621

60

96

282

174

493.74

65.5

67.2

64.6

2379

1332

845

1534

487

60

75

282

158

411.45

81.6

83.7

83.5

1925

1181

865

1060

316

41

49

223

137

342.88

80.7

83.7

83.0

2321

1184

710

1611

474

63

73

327

167

285.73

84.4

85.9

85.1

1966

1119

683

1283

436

50

67

288

164

238.11

77.6

78.5

77.8

2133

1061

682

1451

379

57

58

313

156

198.42

74.9

75.2

74.3

2299

1038

666

1633

372

64

57

345

156

* This test item concentration was not taken into consideration

Table 4 CD54 and CD86 Expression Experiment 2

Sample

Conc.
[μg/mL]

Cell Viability [%]

Mean Fluorescence Intensity

corrected Mean Fluorescence Intensity

Relative Flourescence Intensity (RFI)

Ratio Isotype IgG1 to [%]

CD86

CD54

Isotype

IgG1

CD86

CD54

Isotype IgG1

CD86

CD54

CD86

CD54

C86

CD54

Medium Control

-

95.0

94.3

94.0

3006

1247

645

2361

602

109

96

466

193

Solvent Control

0.20%

94.7

94.5

94.6

2779

1228

604

2175

624

100

100

460

203

DNCB

4.0

78.7

78.7

78.6

8089

2227

582

7507

1645

345

264

1390

383

Test item

710.99*

94.5

94.3

95.5

4945

5314

1099

3846

4215

177

675

450

484

592.49

65.0

60.8

65.3

2527

1523

807

1720

716

79

115

313

189

493.74

67.7

69.0

69.8

2212

1152

765

1447

387

67

62

289

151

411.45

70.3

66.4

77.7

2282

1468

837

1445

631

66

101

273

175

342.88

83.3

86.9

87.5

2587

1045

667

1920

378

88

61

388

157

285.73

87.0

85.0

87.3

1991

1025

732

1259

293

58

47

272

140

238.11

81.2

79.8

79.2

2033

950

615

1418

335

65

54

331

154

198.42

68.2

83.6

79.1

2314

894

605

1709

289

79

46

382

148

* This test item concentration was not taken into consideration

Table 5 Acceptance Criteria

Acceptance Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

cell viability solvent controls [%]

>90

93.1

-

93.9

pass

94.0

-

95.0

pass

number of test dosed with viability >50% CD86

≥4

7*

pass

7*

pass

number of test dosed with viability >50% CD54

≥4

7*

pass

7*

pass

number of test dosed with viability >50% IgG1

≥4

7*

pass

7*

pass

RFI of positive control of CD86

≥150

263

pass

345

pass

RFI of positive control of CD54

≥200

225

pass

264

pass

RFI of solvent control of CD86

<150

111

pass

92

pass

RFI of solvent control of CD54

<200

109

pass

104

pass

MFI ratio CD86/IgG1 for medium control [%]

>105

394

pass

466

pass

MFI ratio CD86/IgG1 for DMSO control [%]

>105

448

pass

460

pass

MFI ratio CD54/IgG1for medium control [%]

>105

176

pass

193

pass

MFI ratio CD54/IgG1for DMSO control [%]

>105

188

pass

203

pass

* One tested dose was not taken into consideration

Table 6 Historical Data

Criterion

mean

SD

N

cell viability solvent controls [%]

97.0

1.3

672

number of test doses with viability >50%

-

-

1786

RFI of positive control of CD86

401.0

146.8

112

RFI of positive control of CD54

576.6

312.0

112

RFI of solvent control of CD86

115.0

15.1

112

RFI of solvent control of CD54

118.8

25.5

112

MFI ratio IgG1/CD86 for medium control [%]

202.4

50.0

112

MFI ratio IgG1/CD86 for DMSO control [%]

221.6

58.5

112

MFI ratio IgG1/CD54 for medium control [%]

141.0

24.7

112

MFI ratio IgG1/CD54 for DMSO control [%]

147.7

25.6

112

Interpretation of results:
other: no activation of dendritic cells
Conclusions:
In this study under the given conditions the test item did not upregulate the expression of the cell surface markers in at least two independent experiment runs. Therefore, the test item might be considered as non-sensitiser. The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.
Executive summary:

A study according OECD TG 442 E was conducted to enable the detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

In the present study the test item was dissolved in DMSO. For the dose finding assay stock solutions with concentrations ranging from 500 mg/mL to 3.91 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.

A CV75 of 592.49 ± 8.15 μg/mL was derived in the dose finding assay. Based on the CV75, the main experiment was performed covering the following concentration steps:

710.99, 592.49, 493.74, 411.45, 342.88, 285.73, 238.11, 198.42 μg/mL

In all experiments no precipitation or turbidity of the test item was observed for all concentration steps when mixing the test item stock solutions with cell culture medium.

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

Clear cytotoxic effects were observed for the highest test item concentration in both experiments and a clear decrease in cell population was observed. The FACS did not show that correctly, therefore, the concentration 710.99 μg/mL was taken out of consideration.

No induction of the expression of the cell surface markers CD86 and CD54 was noted in the other non-cytotoxic concentrations, therefore, the test item can be considered as non-sensitiser.

In this study under the given conditions the test item did not upregulate the expression of the cell surface markers in at least two independent experiment runs. Therefore, the test item is considered as non-sensitiser. The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

For the evaluation of the skin sensitisation potential of the test substance a Weight of Evidence approach was used. The protein reactivity of the test item was assessed in a Direct Peptide Reactivity Assay (DPRA). A Keratinocyte Activation Assay (KeratinoSens) was performed to evaluate the keratinocyte activation potential and a h-CLAT Assay to determine the activation of dendritic cells.

DPRA

A study according OECD TG 442 C was conducted for detection of the sensitising potential of the test item by quantifying its reactivity towards synthetic peptides containing either lysine or cysteine.

The test item was dissolved in dist. water : acetonitrile 1:1 (v/v), based on the results of the pre-experiments. Considering the molecular weight of 872.45 g/mol of the test item, a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently, samples were analysed by HPLC. All test item solutions were freshly prepared immediately prior to use.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (excluding the co-elution control). Samples were centrifuged prior to the HPLC analysis. Since it cannot be determined if the precipitate resulted from the test item or the peptide, the given peak areas and corresponding peptide values can only be considered as an estimation of the peptide depletion.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as not relevant. No co-elution of test item with the cysteine or lysine peptide peak was observed. A possible sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C dist. Water : Acetonitrile 1:1 (v/v)).

The 100 mM stock solution of the test item showed moderate reactivity towards the synthetic peptides. The mean depletion of both peptides was 40.92% and, thus, exceeded the threshold of

6.38%. Even though a precipitate was observed in the cysteine experiment a positive result can still be used. Based on the cysteine 1:10 / lysine 1:50 prediction model the test item can be considered as a skin sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 65.88%. In this study under the given conditions the test item showed moderate reactivity towards both peptides. The test item is considered as skin sensitiser in this in vitro assay. However, the data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP (reference 7.4.1 -1).

KeratinoSens

A study according OECD TG 442 D was conducted which enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

In the present study the test item was dissolved in DMSO. Based on a molecular weight of 872.45 g/mol a stock solution of 200 mM was prepared. Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:

2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98μM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.In the first experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.In the second experiment, a max luciferase activity (Imax) induction of 2.44 was determined at a test item concentration of 7.81μM. The corresponding cell viability was 59.1%. No further significant luciferase induction > 1.5 was found in the tested concentration range. The calculated EC1.5 was <1000μM (5.06μM).No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Under the condition of this study the test item is therefore considered as non sensitiser (reference 7.4.1 -2).

h-CLAT

A study according OECD TG 442 E was conducted to enable the detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

In the present study the test item was dissolved in DMSO. For the dose finding assay stock solutions with concentrations ranging from 500 mg/mL to 3.91 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.

A CV75 of 592.49 ± 8.15 μg/mL was derived in the dose finding assay. Based on the CV75, the main experiment was performed covering the following concentration steps:

710.99, 592.49, 493.74, 411.45, 342.88, 285.73, 238.11, 198.42 μg/mL

In all experiments no precipitation or turbidity of the test item was observed for all concentration steps when mixing the test item stock solutions with cell culture medium.

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

Clear cytotoxic effects were observed for the highest test item concentration in both experiments and a clear decrease in cell population was observed. The FACS did not show that correctly, therefore, the concentration 710.99 μg/mL was taken out of consideration.

No induction of the expression of the cell surface markers CD86 and CD54 was noted in the other non-cytotoxic concentrations, therefore, the test item can be considered as non-sensitiser.

In this study under the given conditions the test item did not upregulate the expression of the cell surface markers in at least two independent experiment runs. Therefore, the test item is considered as non-sensitiser. The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA (reference 7.4.1 -3).

Conclusion:

Based on the results from the in chemico and in vitro studies and considering the Adverse Outcome Pathway (AOP) “2 out of 3” the test item in predicted as non-skin sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

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

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this information, the substance is not considered to be classified for skin sensitisation under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.