1,2-Bis[2-(1,1,2-trifluoro-2-heptafluoropropyloxy-ethylsulfany)-ethoxycarbonyl]-ethanesulfonate sodium salt

Endpoint summary

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

Reference

Reference 1

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

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

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. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

direct peptide reactivity assay (DPRA)

Details on the 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

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion [%]

Value:

3.96

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: lysine run

Parameter:

other: mean peptide depletion [%]

Value:

77.88

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1st experiment

Parameter:

other: mean peptide depleation of both peptides [%]

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).

Reference 2

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

Guideline:

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

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of keratinocytes

Details on the 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

Run / experiment:

other: 1st experiment

Parameter:

other: luciferase activity

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

Run / experiment:

other: 2nd experiment

Parameter:

other: luciferase activity

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

Run / experiment:

other: 2nd experiment

Parameter:

other: EC1.5 [µM]

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.

Reference 3

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

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

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. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of dendritic cells

Details on the 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

Run / experiment:

other: 1st experiment

Parameter:

other: relative fluorescence intensity CD86 [%]

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

Run / experiment:

other: 1st experiment

Parameter:

other: relative fluorescence intensity CD54 [%]

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

Run / experiment:

other: 2nd experiment

Parameter:

other: relative fluorescence intensity CD86 [%]

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

Run / experiment:

other: 2nd experiment

Parameter:

other: relative fluorescence intensity CD54 [%]

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.