[No public or meaningful name is available]

Endpoint summary

Description of key information

Direct Peptide Reactivity Assay (DPRA Test): high chemical reactivity (positive)

in vitro Human Cell Line Activation Test (h-CLAT): positive

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:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Version / remarks:

adopted 14 June 2021

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

SELECTION OF CONCENTRATIONS
The test substance was prepared at a 100 mM concentration considering a purity/contents of 100% and molecular weight of 532.42 g/mol. The C-containing peptide was incubated with the test substance in a ratio of 1:10 (0.5 mM peptide, 5 mM test substance) and the K-containing peptide in a ratio of 1:50 (0.5 mM peptide, 25 mM test substance).

MATERIAL AND TECHNICAL EQUIPMENT
DPRA:
HPLC: Liquid chromatograph Thermo Scientific, Dionex Ultimate 3000
consisting of the following modules:
Pump: HPG-3400RS
Autosampler: WPS-3000TSL
Column oven: TCC-3000
UV-Detector: DAD-3000
Column: ZORBAX SB-C18 2.1 x 100 mm, 3.5 µm with guard column
SecurityGuard Ultra Cartridges, UHPLC C18 for 4.6 mm ID
(Phenomenex)
HPLC mobile phase A: H2O/ACN/TFA 950/50/1 V/V/V
HPLC mobile phase B: ACN/H2O/TFA 950/50/0.85 V/V/V
Incubator: Thermo Scientific; for incubation at 25°C ± 2.5°C
pH meter: Readability +/- 0.1 pH units.
For adjusting pH-values of buffers.
Reagents for preparing
the buffers (Sigma): for pH 7.5 phosphate buffer (used for solving C-containing peptide):
Sodium phosphate, monobasic monohydrate, CAS no. 10049-21-5
Sodium phosphate, dibasic heptahydrate, CAS no. 7782-85-6
for pH 10.2 ammonium acetate buffer (used for solving Kcontaining peptide):
Ammonium acetate, CAS no. 631-61-8
Ammonium hydroxide, 28% – 30%, CAS no. 1336-21-6
CONTROLS
Vehicle control (VC): acetonitrile
Positive control (PC): Ethylene glycol dimethacrylate (EGDMA; CAS-no. 97-90-5),
prepared as a 50 mM solution in acetonitrile.
Co-elution control (SK): Sample prepared of the respective peptide buffer and the test
substance but without peptide.
TEST-SUBSTANCE PREPARATIONS
The test-substance preparations were prepared on a weight per volume basis within 4 hours
of preparation of test-substance samples.1
The test substance was prepared as a 100 mM preparation in acetonitrile considering a
molecular weight of 532.42 g/mol and a purity/contents of 100%. After short stirring the test
substance was soluble in the vehicle.
Vehicle: acetonitrile
Reason for the vehicle: The test substance was soluble in acetonitrile.
ANALYSES
Because the test-substance preparation was incubated with the peptide shortly after
preparation, no analysis of the test substance in the vehicle was performed.
EXPERIMENTAL PROCEDURE
The test substance was dissolved in a suitable vehicle. Per run three samples of the test
substance were incubated with each peptide. Additionally, triplicates of the concurrent vehicle
control (= VC) were incubated with the peptides. The remaining non-depleted peptide
concentration was determined thereafter by HPLC with gradient elution and UV-detection at
220 nm.
In addition, calibration samples of known peptide concentration, prepared from the respective
peptide stock solution used for test-substance incubation, were measured in parallel with the
same analytical method.
Test substance solubility
Prior to the assay the solubility of the test substance at a concentration of 100 mM was tested.
A suitable non-reactive, water-miscible solvent which dissolves the test substance completely
(no visible precipitation or cloudyness of the test-susbtance preparation) should be used. The
preferred solvent was acetonitrile. When not soluble in acetonitrile solutions in water,
isopropanol, acetone, propanol, methanol or mixtures of these solvents were tried.
Preparation of peptide stock solutions
Peptide stock solutions in a concentration of 0.667 mM were prepared in pH 7.5 phosphate
buffer (C-containing peptide) or pH 10.2 ammonium acetate buffer (K-containing peptide). The
peptide stock solution was used for preparing the calibration samples and the test substance
and control samples.
Preparation of calibration samples
The following calibration samples were prepared from the peptide stock solutions in 20%
acetonitrile in the respective buffer (= dilution buffer) using serial dilution:: Calibration samples of DPRA.
Calib. 1 Calib. 2 Calib. 3 Calib. 4 Calib. 5 Calib. 6 Dilution buffer
mM peptide 0.534 0.267 0.134 0.067 0.033 0.017 0.000
The analysis of the calibration samples was started before analysis of the test-substance
samples.

Preparation of the test-substance samples
The samples were prepared in triplicates for each peptide according to the pipetting scheme
given below.
Preparation scheme of samples.
C-peptide
750 µL C-peptide stock solution
200 µL solvent (vehicle)
50 µL test-substance preparation
(or PC-preparation or solvent (VC))
K-peptide
750 µL K-peptide stock solution
250 µL test-substance preparation
(or PC-preparation or solvent (VC))
The samples were prepared in suitable tubes, capped tightly and incubated at 25°C ± 2.5°C in
the dark for 24 +/- 2 hours. Visual inspection for solubility was performed directly after sample
preparation and prior to HPLC analysis. Unsolved samples were centrifuged and/or filtrated
prior to injection into the HPLC in order to remove any unsolved particles. The HLPC analysis
of the batch of samples started about 24 hours after sample preparation and the analysis time
itself did not exceed 30 hours, with exception of the second set B of vehicle controls of the K-containing peptide in the 2nd test run.
Preparation of the vehicle controls
Several vehicle controls were prepared in triplicates in the same way as the test-substance
samples described above but with the vehicle (acetonitrile) instead of the test substance: One
set (set A) was analyzed together with the calibration samples without incubation and serves
as a performance control. Another three sets (two sets B and set C) were prepared and
incubated with the samples. Sets B were placed at the very start and ending of the sample list
and serve as stability control of the peptide over the analysis time. Set C was analyzed with
the samples and serves for calculation of the peptide depletion of any chemical formulated in
the vehicle.
Preparation of the co-elution control
One sample per peptide was prepared in the same way as the test-substance samples
described above but without the peptides. Instead the respective peptide buffer was used. The
samples were analyzed together with the calibration samples. Samples which were visually
turbid or display precipitates were centrifuged and/or filtrated prior to injection into the HPLC in
order to remove any unsolved particles.
Measurement of peptide concentrations
The analyses of the samples were performed via HPLC under the following conditions:
Table 3: HPLC conditions used for analysis of DPRA samples.
Column: ZORBAX SB-C18 2.1 x 100 mm, 3.5 µm with guard column
SecurityGuard Ultra Cartridges, UHPLC C18 for 4.6 mm ID
(Phenomenex)
Mobile phase: A: H2O/ACN/TFA 950/50/1 V/V/V
B: ACN/H2O/TFA 950/50/0.85 V/V/V
Flow: 0.50 mL/min (standard analytic method)
0.35 mL/min (alternative analytic method)
Gradient of
standard
analytic method:
time [min] %B
0 5
8 20
8.1 90
10 90
10.1 5
16 5

Gradient of
alternative
analytic method:
time [min] %B
0 5
0.5 5
10.5 25
11 90
13 90
13.5 5
20 5
Wavelength: 220 nm and 258 nm
Injection volume: 2 µL
Software: Dionex Chromeleon

DATA EVALUATION
Table(s) and/or figure(s) of measured parameters presented in the report were produced using
PC based tabular calculation software. The mean and individual data are not always rounded
but the significant digits are produced by changing the display format. As a consequence,
calculation of mean values using the individual data presented in the report will, in some
instances, yield minor variations in value.
The integrated peak areas were transferred electronically into EXCEL data spreadsheets to
carry out the necessary calculations.
Some test substances or reaction products may co-eluate with the peptides. In these cases
where proper integration and calculation of peptide depletion was not possible, the result for
the respective peptide is reported as interference.
For evaluation of peptide depletions peak areas at 220 nm are used. When samples were
additionally analyzed by measuring UV absorbance at 258 nm, the area ratio 220 nm/ 258 nm
may be calculated and serve as a measure of peak purity. The ratio of a pure peptide peak
should be consistent over all samples (100% ± 10% of the mean of the vehicle controls).
However, due to small peak areas calculation of the area ratio may not be possible for all
samples.

Calculation of the peptide concentrations
For each peptide and test run a calibration curve is generated from the measured peak areas
of the calibration samples of known peptide concentration.
The peptide concentration of the samples is calculated with the respective calibration curve
using linear regression (b = axis intercept; m = slope).
Peptide conc. [mM] = (peak area at 220 nm [mAU x s] – b) / m

Calculation of the peptide depletion
The peptide depletion of a sample is calculated as follows:
Peptide depletion of a sample = (1 – (peptide conc. sample [mM] / mean peptide conc. VC [mM]) x 100 [%]
The mean peptide depletion for each of the two peptides is calculated as the mean value of
the three samples conducted for each peptide and test substance (C-containing and K-containing peptide depletion; example calculation for C-containing peptide):
C-cont. peptide depletion of a test substance [%] = mean [C-cont. peptide depletion of samples 1 – 3] [%]
When a negative value for C- or K-containing peptide depletion is obtained the value is
considered zero for calculation of the mean peptide depletion.
The mean peptide depletion of a test substance is calculated as the mean value of C-containing
peptide depletion and K-containing peptide depletion:
Mean peptide depletion [%] = (C-cont. peptide depletion [%] + K-cont. peptide depletion [%]) / 2

ACCEPTANCE CRITERIA
If any of the acceptance criteria mentioned below is not met, repetition of the test is considered.
In the case of an unambiguous result the study director may decide that an experiment is valid
despite a deviation of acceptance criteria. However, reasonable justification must then be
given.
The standard calibration curve should have an r² >0.99.
The vehicle control samples of sets A and C should be 0.50 mM +/- 0.05 mM.
The CV of the nine vehicle controls B and C should be < 15%.
Since the mean peptide depletion for each peptide is determined from the mean of three single
samples, the variability between these samples should be acceptably low (SD < 14.9% for %
cysteine depletion and < 11.6% for % lysine depletion).
In addition the positive control should cause depletion of both peptides comparable to historic
data.

EVALUATION OF RESULTS
Peptide depletion
Chemical reactivity was determined by mean peptide depletion [%] and was rated as high,
moderate, low, or minimal:
Evaluation criteria of DPRA; cysteine 1:10 / lysine 1:50 prediction model.
Mean peptide depletion
[%] Reactivity Evaluation
> 42.47 high reactivity positive
> 22.62 ≤ 42.47 moderate reactivity positive
> 6.38 ≤ 22.62 low reactivity positive
≤ 6.38 minimal or no reactivity negative
In addition to the evaluation criteria cited in the table above the following borderline range
applies: 4.95% - 8.32%. Results in this range are evaluated “inconclusive”.
In the case mean peptide depletion [%] cannot be determined due to invalid K-peptide depletion
(e.g. insolubility of the K-peptide samples or interference in the samples of the K-peptide) but
valid C-peptide depletion is available, evaluation is performed as follows:
Evaluation criteria of DPRA; cysteine 1:10 prediction model.
C peptide depletion [%] Reactivity Evaluation
> 98.24 high reactivity positive
> 23.09 ≤ 98.24 moderate reactivity positive
> 13.89 ≤ 23.09 low reactivity positive
≤ 13.89 minimal or no reactivity negative
In addition to the evaluation criteria cited in the table above the following borderline range
applies: 10.56% - 18.47%. Results in this range are evaluated “inconclusive”.
A single test run should be sufficient for a test substance when the result is unequivocal.
However, in cases of results close to the threshold used to discriminate between positive and
negative results (i.e. mean percent depletion falls in the range of 3% to 10% for the cysteine
1:10/lysine 1:50 prediction model or cysteine percent depletion falls in the range of 9% to 17%
for the cysteine 1:10 prediction model), additional testing is performed. A second run is
conducted in these cases, as well as a third one in case of discordant results between the first
two runs.

Limitations of the evaluation by insolubility and gravimetric procedure
For test substances that are not completely soluble by visual observation in the sample
preparations containing the peptides immediately after preparation or after 24 hours, or when
a gravimetric procedure is applied (with the exception of application of the undiluted test
substance (liquids) or the maximal soluble test-substance concentration (solids)), the result
may be under-predictive due to limited availablity of the test substance. In this case mean
peptide reactivity ≤ 6.38% (cysteine 1:10 / lysine 1:50 prediction model) or ≤ 13.89% (cysteine
1:10 prediction model) is interpreted as “inconclusive”. However, a mean peptide depletion >
6.38% or > 13.89% is considered as “positive”.

HISTORIC CONTROL DATA
Historic control values of negative and positive controls, gathered over an appropriate time
period, are presented in section 4.6. These data demonstrate the reproducibility of results and
robustness of the procedures. They are used to derive suitable acceptance criteria (see section
3.9) for the test system.

Vehicle / solvent:

acetonitrile

Positive control:

other: Ethylene glycol dimethacrylate (EGDMA; CAS-no. 97-90-5)

Group:

test chemical

Run / experiment:

mean

Parameter:

cysteine depletion

Value:

100 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Group:

other: positive control

Run / experiment:

mean

Parameter:

cysteine depletion

Value:

65.73 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Group:

test chemical

Run / experiment:

other: mean 1st test run

Parameter:

lysine depletion

Value:

23.45 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Group:

test chemical

Run / experiment:

other: mean 2nd test run

Parameter:

lysine depletion

Value:

5.68 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Group:

other: positive control

Run / experiment:

other: mean 2st test run

Parameter:

lysine depletion

Value:

23.45 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Group:

other: positive contriol

Run / experiment:

other: mean 2nd test run

Parameter:

mean cystein depletion

Value:

12.33 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Outcome of the prediction model:

high reactivity [in chemico]

Other effects / acceptance of results:

CYSTEINE-CONTAINING PEPTIDE
Results of the 1st test run are not included in the report since the test run was invalid (mean
depletion of the vehicle control samples of set C was not within the range 0.50 mM +/- 0.05
mM).

Cysteine-peptide calibration curve
The calibration curve of the cysteine-containing peptide shows the following parameters:
Slope: 9.5
Axis intercept: 0.163
Correlation: 0.99792

Cysteine-peptide vehicle controls in acetonitrile
The mean peptide concentration of the three samples of set A was calculated to be
0.496 mM with a SD of 0.002 mM, demonstrating good performance.
The mean peptide concentration of the three samples of set B, analyzed at the beginning of
the sample list was calculated to be 0.464 mM with a SD of 0.004 mM. The other three samples
of set B, analyzed at the end of the sample list had a mean peptide concentration of
0.425 mM with a SD of 0.007 mM.
The CV of the 9 vehicle control samples of sets B and C was calculated to be 3.9%. Thus the
peptide was considered stable over the time of analysis.

LYSINE-CONTAINING PEPTIDE

Lysine-peptide calibration curve
The calibration curves of the lysine-containing peptide show the following parameters:
Test run 1:
Slope: 14.9
Axis intercept: 0.036
Correlation: 0.99996
Test run 2:
Slope: 22.1
Axis intercept: 0.026
Correlation: 0.99996
4.2.1.1. Lysine-peptide vehicle controls in acetonitrile
1st test run:
The mean peptide concentration of the three samples of set A was calculated to be
0.501 mM with a SD of 0.004 mM, demonstrating good performance.
The mean peptide concentration of the three samples of set B, analyzed at the beginning of
the sample list was calculated to be 0.506 mM with a SD of 0.000 mM. The other three samples
of set B, analyzed at the end of the sample list had a mean peptide concentration of
0.510 mM with a SD of 0.003 mM.
The CV of the 9 vehicle control samples of sets B and C was calculated to be 0.4%. Thus the
peptide was considered stable over the time of analysis.
2nd test run:
The mean peptide concentration of the three samples of set A was calculated to be
0.516 mM with a SD of 0.008 mM, demonstrating good performance.
The mean peptide concentration of the three samples of set B, analyzed at the beginning of
the sample list was calculated to be 0.511 mM with a SD of 0.002 mM. The other three samples
of set B, analyzed at the end of the sample list had a mean peptide concentration of
0.492 mM with a SD of 0.001 mM. These last three samples were analyzed later than 30 hours
after beginning of the analysis. Since stability was still given, this deviation did not impair
validity of the sstudy.
The CV of the 9 vehicle control samples of sets B and C was calculated to be 1.8%. Thus the
peptide was considered stable over the time of analysis.

SOLUBILITY OF THE TEST-SUBSTANCE SAMPLES WITH THE PEPTIDES
The test substance was dissolved in acetonitrile. The samples of the test substance with the
peptides were solutions. Visual observation after the 24-hour incubation time did not reveal
precipitates in any samples of the test substance with the peptides.
CO-ELUTION
Co-elution of the test substance and the K-containing peptide occurred using two different
analysis methods as demonstrated by the co-elution control (see sample chromatograms in
the Appendix) and peak purity (for values of area ratio 220 nm/258 nm see section 4.2.2). Thus,
a meaningful K-peptide depletion could not be determined.
No co-elution of the test substance and C-containing peptide occurred.

Relevant tables are attached as background material.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Based on the observed results and applying the cysteine 1:10 prediction model it was concluded that Emuldur 3643 shows high chemical reactivity in the DPRA under the test conditions chosen.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes

Type of study:

human Cell Line Activation Test (h-CLAT)

Details of test system:

THP-1 cell line [442E]

Details on the study design:

An alternative method has been developed for the evaluation of the skin sensitisation potential by measuring phenotypic changes, such as CD86 and CD54 expression on dendritic cells. The human leukemia cell line THP-1 is used as a surrogate for human myeloid dendritic cells, since these cells also show enhanced CD86 and/or CD54 expression when treated with sensitisers.
The purpose of the Human Cell Line Activation Test (h-CLAT) is to assess the skin sensitising potential of Emuldur 3643 in an appropriate solvent (DMSO, saline or culture medium) when administered to THP-1 cells for 24 hours. The test item concentrations for the main experiment (h-CLAT) of Emuldur 3643 are determined by cytotoxicity tests.
This human cell line activation test can be used as part of a testing battery (including e.g. DPRA (Direct Peptide Reactivity Assay), ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals.
The technical proficiency of the h-CLAT with the OECD 442E guideline recommended proficiency substances was demonstrated.

Controls for Cytotoxicity Test and h-CLAT
Concurrent controls were used for several ICCR-Roßdorf GmbH studies performed simultaneously.

Medium Control
Name: Culture medium

Solvent Control for the Test Item
Name: DMSO (final concentration for cytotoxicity and for h-CLAT 0.2%)

Positive Control (h-CLAT)
Name: DNCB (2,4-dinitrochlorobenzene, CAS No.: 97-00-7) final concentration: 3 and 4 μg/mL, Purity ≥ 99%)
Solvent: DMSO (final concentration 0.2%)

Solvent Control for the Positive Control (h-CLAT)
Name: DMSO (Dimethyl sulfoxide) in culture medium, final concentration 0.2%, Purity ≥ 99%

TEST ITEM PREPARATION
On the day of the experiment (prior to start) Emuldur 3643 was dissolved in DMSO and further diluted in culture medium.
As highest test item concentration 1000 μg/mL (0.2% (v/v) DMSO in culture medium) was used in the cytotoxicity test as recommended by the OECD 442E guideline. For the cytotoxicity test eight concentrations of the test item were analysed. For this, dilutions were prepared by 1:2 serial dilutions.
With reference to the CV75 parameter the highest tested concentration in the main experiments was 31.2 μg/mL (stock solution of 15.6 mM).

TEST SYSTEM AND SUPPORTING INFORMATION
Reasons for the Choice of THP-1 Cells
THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC, #TIB-202. THP-1 cells are used as a surrogate for human myeloid dendritic cells, because the THP-1 cells also show enhanced CD86 and/or CD54 expression when treated with sensitisers.

THP-1 Cell Cultures
Stocks of the THP-1 cell line are stored in liquid nitrogen in the cell bank of ICCR-Roßdorf GmbH (aliquots of cells in freezing medium at 1 × 106 to 2 × 106 cells/mL) allowing the repeated use of the same working cell stock in experiments. Therefore, the parameters of the experiments remain similar, because of the reproducible characteristics of the cells. Thawed stock cultures are propagated at 37 ± 1.5 °C in plastic flasks. The cells are sub-cultured at least twice a week. The cell density should not exceed 1 × 106 cells/mL. The THP-1 cell suspension is incubated at 37 ± 1.5 °C and 5.0 ± 0.5 % carbon dioxide atmosphere. Cells can be used up to two months after thawing (passage number should not exceed 30).
The passage numbers of the used THP-1 cells were 21/culture 1 and 21/culture 2 in the cytotoxicity tests and 12 and 14 in the h-CLAT for runs 1 and 2, respectively.

Culture Medium
RPMI 1640 Medium, GlutaMAXTM Supplement including 25 mM HEPES, supplemented with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1 mM sodium pyruvate and appropriate antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin) is used to culture the cells during the assay. Medium with supplements has to be stored at 2 - 8 °C and used within one month.

Preparation and Seeding of THP-1 Cells
On the day of the cytotoxicity or main experiment (h-CLAT) directly before the treatment of the cells, a volume of 500 μL with a cell density of 1.8 - 2 × 106 THP-1 cells/mL was seeded in each corresponding well of a 24-well flat bottom plate.

Experimental Design and Procedures of the Cytotoxicity Test
Dose Finding Assay (Flow cytometer)
The test item concentrations investigated in the main experiment (h-CLAT) were determined with two cytotoxicity tests. The tests were performed with independent cell cultures. The test item was prepared separately for each run.

Treatment of the Cells
The test item dilutions were prepared freshly before each experiment.
Each DMSO solution was diluted with culture medium before application of the test solution to the cells to reach a final concentration of 0.2% (v/v) in the culture medium.
Each volume (500 μL) of the dilutions of the test item, culture medium and solvent control (0.2% (v/v) DMSO in culture medium) was added to the cells.
The treated THP-1 cells were incubated for 24 ± 0.5 hours. All dose groups were tested in one replicate for each cytotoxicity test. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations.
Each concentration of the test item, culture medium and solvent control were prepared for the 7-AAD staining.

Staining of the Cells
Each test item-treated and not test item-treated cells were collected in sample tubes, centrifuged (approx. 250 × g, 5 min), washed twice (2 - 8 °C) with 2 mL FACS buffer (PBS with 0.1% (w/v) BSA) and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 μL of a 7-AAD solution were added in each sample tube.

Flow Cytometry Acquisition (Cytotoxicity Test)
Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions.
The cytotoxicity was analysed by flow cytometry using the software Cellquest Pro 6.0. The 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD fluorescence signal.

Preparation of the acquisition (Cytotoxicity Test)
The following acquisition plots were prepared:
• 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
• Histogram plot of the FL-3 channel
The voltage of FSC and SSC was set to appropriate levels. FSC and SSC are not needed for the analysis, but the FSC/SSC plot should be checked to make sure that a single population appears without contamination or excessive debris. The FL-3 voltage was set and compensate to appropriate position (FACSCalibur, Becton Dickinson GmbH).
The cell viability was measured by gating-out dead cells stained with 7-AAD. A total of 10,000 living cells were analysed.
The maintenance of the flow cytometer was in accordance with the manufacturer’s instructions. The process of washing was conducted very carefully since insoluble chemicals could flow in the flow line.

Flow Cytometry Analysis (Cytotoxicity Test)
The cell viability is shown by the cytometry analysis program (% total) and is calculated according to the following equation: Cell Viability [%]= (Number of living cells)/(Total Number of acquired cells) ×100
The CV75 value, a concentration showing 75% of THP-1 cell survival (25% cytotoxicity), is calculated by log-linear interpolation.

Acceptability of the Cytotoxicity Assay
The cytotoxicity test is considered to be acceptable if it meets the following criteria:
• The cell viability of the medium and solvent control should be more than 90%.

Calculation of the Test Doses for the Main Experiment (h-CLAT)
The mean of the two CV75 values was used to determine the dose-range for the main experiment (h-CLAT). Eight final concentrations (μg/mL) were used for the test item in the main experiment (h CLAT). The highest concentration used was 1.2 × mean CV75 with a serial dilution factor of 1.2.

Experimental Design and Procedures of h-CLAT
The test item was tested in two independent runs. The tests were performed with independent cell cultures (cells are collected from different culture flasks). The test item was prepared separately for each run.
Treatment of the Cells
The test item dilutions were prepared freshly before each experiment.
For the test item exposure, the highest dose solution calculated from the cytotoxicity test was prepared corresponding to 1.2 × CV75. Further 7 dilutions were prepared by serial 1:1.2 dilution. Each solution was diluted with culture medium before application of the test solution to the cells to reach a final concentration of 0.2% (v/v) DMSO in the medium.
Each volume (500 μL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations.
Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).

Staining of the Cells
The triplicates of each test item-treated and not test item-treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250 × g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended and blocked with 600 μL of blocking solution at 2 - 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged, and the cell pellets were re-suspended in 100 μL FACS buffer. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control).
All solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis procedures.
The cells with the different antibodies or the IgG1 were mixed and incubated light protected for 30 ± 5 min. at 2 - 8 °C (on ice).

Sample Preparation for Measurement
After staining with the antibodies, the cells were washed twice (2 - 8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 μL of a 7-AAD solution were added.

Flow Cytometry Acquisition
Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions.
The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry using the software Cellquest Pro 6.0. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD fluorescence signal.

Preparation of the acquisition
The following acquisition plots were prepared:
• 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
• Histogram plot of each channel (FL-1 and FL-3, respectively)
The voltage of FSC and SSC was set with untreated cells to appropriate levels. FSC and SSC are not needed for the analysis, but the FSC/SSC plot was checked to make sure that a single population appears without contamination or excessive debris. The FL-1 and FL-3 voltage were set and compensate to appropriate position. The FL-1 voltage was set using the FITC labelled-mouse IgG1 medium-treated cells tube, as such the MFI of control cells was set in the range between 1.0 and 4.0 (Geo Mean) and in the range between 3.0 and 4.0 (Geo Mean) with the FITC labelled CD54 medium-treated cells (FACSCalibur, Becton Dickinson GmbH).
The cell viability was detected by setting an R1-gate (dead cells are gated-out by staining with 7-AAD). Therefore, the R1 gate was set approximately at the middle position between the peak of the negative fraction and the positive fraction in the FL-3 histogram using DNCB-treated cells. The negative fraction corresponds to the living cells and was kept for the subsequent analyses. For each control and all test item concentrations, the cell viability was recorded from the isotype control cell tube (stained with FITC labelled-mouse IgG1), the CD54 and CD86 cell tube, where only the isotype control cells were used for the cell viability evaluation.
The maintenance of the flow cytometer was in accordance with the manufacturer’s instructions. The process of washing was conducted very carefully since insoluble chemicals could flow into the flow line.
Acquisition
Dead cells were gated-out by staining with 7-AAD. Gating by FSC (forward scatter) and SSC (side scatter) was not done. A total of 10,000 living cells were analysed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. The other tubes were acquired without changing the settings of the cytometer. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) was calculated, but excluded from the evaluation, if the cell viability was less than 50% (due to diffuse labelling of cytoplasmic structures that could be generated due to cell membrane destruction).

Data Analysis and Interpretation
Flow Cytometry Analysis
The RFI is used as an indicator of CD86 and CD54 expression, and is calculated for each concentration of every chemical.
The cell viability from the isotype control cells, CD54 and CD86 cells is calculated according to the following equation: Cell Viability [%]= (Number of living cells)/(Total Number of acquired cells) ×100
where the mean of the three samples is used for the cell viability evaluation.

Calculation of EC150 and EC200
For test items predicted as positive with the h-CLAT, optionally, two effective concentrations (EC) values, the EC150 for CD86 and EC200 for CD54, i.e. the concentration at which the test chemicals induced a RFI of 150 or 200, are determined, if possible. These EC values potentially could contribute to the assessment of sensitising potency when used in integrated approaches such as IATA.

Acceptance Criteria
The following acceptance criteria should be met when using the h-CLAT method:
• Cell viability of medium control and DMSO control should be more than 90%.
• In the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
• For both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
• In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50% in at least one concentration of the two tested positive control concentrations.
• For the test item, the cell viability should be more than 50% in at least four tested concentrations in each run.
Negative results are acceptable only for test items exhibiting a cell viability of < 90% at the highest concentration tested (i.e. 1.2 × CV75). If the cell viability at 1.2 × CV75 is ≥ 90% the negative result should be discarded. In such case it is recommended to try to refine the dose selection by repeating the CV75 determination. It should be noted that when 5000 μg/mL in saline (or medium or other solvents/vehicles), 1000 μg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test chemical, a negative result is acceptable even if the cell viability is > 90% (OECD 442E guideline).

Prediction model
For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs (OECD 442E guideline):
− The RFI of CD86 is ≥ 150% at any tested concentration (with cell viability ≥ 50%);
− The RFI of CD54 is ≥ 200% at any tested concentration (with cell viability ≥ 50%).
Otherwise, the h-CLAT prediction is considered NEGATIVE (see chapter 5.6.8.1).
Based on the above, if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered POSITIVE and a third run does not need to be conducted. Similarly, if the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE without the need for a third run. If, however, the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 (hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third run is required. If this third run is negative for both markers (hereinafter referred to as N), the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered POSITIVE. An h-CLAT prediction should be considered in the framework of an IATA (OECD 442E guideline).

MAJOR COMPUTERIZED SYSTEMS
The cytotoxicity and the expression of cell surface antigens (CD54, CD86) was analysed using the flow cytometer FACSCalibur, Becton Dickinson GmbH, software Cellquest Pro 6.0.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Group:

test chemical

Run / experiment:

mean

Parameter:

CV75 [442D and 442E]

Value:

26.02 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

RFI CD86>200 [442E]

Remarks:

EC200

Value:

19.1 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Outcome of the prediction model:

positive [in vitro/in chemico]

Discussion

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of Emuldur 3643 dissolved in DMSO when administered to THP-1 cells for 24 ± 0.5 hours.

This human cell line activation test can be used as part of a testing battery (including e.g. DPRA (Direct Peptide Reactivity Assay), ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals.

The highest test item concentration for the main experiment (h-CLAT) of Emuldur 3643 was previously determined by two cytotoxicity tests.

The highest concentration of the test item in both dose finding assays was 1000 μg/mL in DMSO in accordance to the OECD Guideline 442E. Cytotoxic effects (threshold of cytotoxicity: < 75%) were observed following incubation with the test item starting with the concentration of 31.3 μg/mL up to the highest tested concentration (1000 μg/mL) in both cytotoxicity tests. The mean CV75 value of both cytotoxicity tests was calculated as 26.02 μg/mL.

The following concentrations of the test item were tested in the main experiments (h-CLAT):

8.71, 10.4, 12.5, 15.0, 18.1, 21.7, 26.0, 31.2 μg/mL

The test item with a log Pow of 0.55-3.60 was tested in 2 independent runs. The RFI of CD86 and/or CD54 was equal or greater than 150% and 200%, respectively, in at least one concentration of both runs. Therefore, the h-CLAT prediction is considered positive for the tested test item in this h-CLAT.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test item Emuldur 3643 with a log Pow of 0.55-3.60 activated THP-1 cells under the test conditions of this study. Therefore, the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Direct Peptide Reactivity Assay (DPRA)

The reactivity of the test substance towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA) according to OECD 442C guideline and GLP. For this purpose, the test substance was incubated with synthetic peptides for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm. The test substance was dissolved at 100 mM in acetonitrile. Two test runs were performed. Per test run three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50 (for K-containing peptide). Additionally, triplicates of the concurrent vehicle control (= VC) were incubated with the peptides. Further, in order to detect possible interference of the test substance with the peptides, a co-elution control was performed and the samples were analyzed by measuring UV absorbance at 258 nm in order to calculate the area ratio 220 nm / 258 nm.

The following results were obtained in the DPRA:

The test substance was dissolved in acetonitrile at a concentration of 100 mM. The samples of the test substance with the peptides were solutions at the time of preparation. Visual observation after the 24-hour incubation time did not reveal precipitates in any samples of the test substance with the peptides.

C-peptide:

Two test runs were performed since validity criteria were not met in the first test run. The second test run met all validity criteria. No co-elution of test substance and the C-peptide was present using the standard analysis method.

K-peptide:

Two test runs were performed since co-elution of the test substance and the K-peptide was observed in the first test run. In the second test run, co-elution was still noticed using an alternative analysis method. The C-peptide depletion, caused by the test substance was determined to be 100.00%. A meaningful K-peptide depletion could not be determined due to co-elution.

Due to the interference observed in the K-peptide samples calculation of mean peptide depletion is not applicable and the cysteine 1:10 prediction model is used for evaluation.

Based on the observed results and applying the cysteine 1:10 prediction model it was concluded that test substance shows high chemical reactivity in the DPRA under the test conditions chosen.

in vitro Human Cell Line Activation Test (h-CLAT)

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of the test substance dissolved in DMSO when administered to THP-1 cells for 24 ± 0.5 hours. The study was performed in accordance with OECD guideline 442E and GLP.

This human cell line activation test can be used as part of a testing battery (including e.g. DPRA (Direct Peptide Reactivity Assay), ARE-Nrf2 (luciferase test method)) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals.

The highest concentration of the test item in both dose finding assays was 1000 μg/mL in accordance to the OECD Guideline 442E.

Cytotoxic effects (threshold of cytotoxicity: < 75%) were observed following incubation with the test item starting with the concentration of 31.3 μg/mL up to the highest tested concentration (1000 μg/mL) in both cytotoxicity tests. The mean CV75 value of both cytotoxicity tests was calculated as 26.02 μg/mL.

The following concentrations of the test item were tested the main experiments (h-CLAT):

8.71, 10.4, 12.5, 15.0, 18.1, 21.7, 26.0, 31.2 μg/mL

The test item with a log Pow of 0.55-3.60 was tested in 2 independent runs. The relative fluorescence intensity (RFI) of CD86 and/or CD54 was equal or greater than 150% and 200%, respectively, in at least one concentration of both runs. Therefore, the h-CLAT prediction is considered positive for the test item in this h-CLAT.

All acceptance criteria were met. The cell viability of the medium and DMSO control was > 90%. For both medium and solvent control, the MFI ratio of CD86 and CD54 to isotype control was > 105%. In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was > 50%. For the test item, the cell viability was more than 50% in at least four tested concentrations in each run.

In conclusion, the test item with a log Pow of 0.55-3.60 activated THP-1 cells under the test conditions of this study. Therefore, the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

LuSens

This in vitro Skin Sensitisation Test ARE-Nrf2 Luciferase Test Method (LuSens) was performed to assess the inflammatory responses in the keratinocytes as changes in gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways (second key event of the skin sensitization AOP) of Emuldur 3643.

In the first dose finding assay, cytotoxic effects were observed following incubation with the test item starting with the concentration of 3.91 μM up to the highest tested concentration of 2000 μM (threshold of cytotoxicity: <70% cell viability). The CV75 value of the cytotoxicity test was calculated as 2.1 μM.

The test item was tested in 2 independent main experiments. All tested concentrations (1.01-2.5 μM) resulted from the CV75 obtained in the first dose finding assay showed cytotoxic effects. The second main experiment was invalid due to low absorption values and will not be reported. Therefore, a confirmatory second dose finding assay was performed. Here. the CV75 was 1.9 μM. In a third dose finding assay the test concentrations were adjusted with 10.0 μM as top dose and a dilution factor of 1.3 to better characterise the cytotoxicity. The resulting CV75 was 3.7 μM and confirmed the cytotoxicity of the test item.

The acceptance criteria were met, only the cytotoxicity criterion was not met:

- The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥2.5 (ME 1: 6.04).

- The positive control had a relative cell viability ≥70% as compared to the solvent control (ME 1: 98.76%).

- The average luciferase activity induction obtained with the negative control, 5000 μM lactic acid (ME 1: 1.17), as well as the basal expression of untreated cells was < 1.5-fold as compared to the average solvent control (ME 1: 1.08).

- The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was below 20% in the main experiment (ME 1: 7.2%).

- All test item concentrations had a cell viability of <70% relative to the solvent control. Therefore, the test item is not suitable for the LuSens assay.

In conclusion, the test item Emuldur 3643 is not suitable for the LuSens assay due to the high cytotoxicity. Upon sponsor’s request the study was cancelled.

Justification for classification or non-classification

The test substance is peptide reactive and activates dendritic cells. The potential of the test substancew to activate keratinocytes was not conclusively evaluated due to high cytotoxicity (test material not suitable for the LuSens assay). Based on the results of the DPRA and h-CLAT, the test material is predicted to be a skin sensitizer. As a result the substance is classified as skin sensitizer Cat. 1 under Regulation (EC) No 1272/2008.