[No public or meaningful name is available]

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Product of 1,1,1-Trimethylol propane triacrylate with aziridine and 1-cyclooctyl-1,4-diazocane and 4-hydroxy-4-methylpentan-2-one

Molecular formula:

C6H12N2 - C47H77O16N4

IUPAC Name:

Reaction Product of 1,1,1-Trimethylol propane triacrylate with aziridine and 1-cyclooctyl-1,4-diazocane and 4-hydroxy-4-methylpentan-2-one

Test material form:

liquid

Details on test material:

Name of test item: Emuldur 3643
Test item No.: 19/0071-2
Batch identification: 27771924U0
Purity: 100 % (UVCB*, for details see analytical characterization No.20L00150) *Substances of unknown or variable composition, complex reaction products.
Homogeneity: The test item was homogeneous by visual inspection.
Storage stability: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
Expiry date: July 15, 2021
Storage conditions: Ambient (RT)
Physical state / color: Liquid / colorless to yellowish, clear

In chemico test system

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)

Results and discussion

In vitro / in chemico

Results

Results 1

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

Results 2

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

Results 3

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

Results 4

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

Results 5

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

Results 6

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.

Applicant's summary and conclusion

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.