[No public or meaningful name is available]

Endpoint summary

Description of key information

Skin:

The mean of the SPCC and SPCL depletion was 1.3% and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

The test item is classified as inconclusive in the KeratinoSens assay.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 26 Mar to 16 Apr 2021

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 KeratinoSens™ test method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Specific details on test material used for the study:

Batch No.: 465884
Purity: 100%

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution
The test item was dissolved in DMSO to a final concentration of 200 mM (orange solution). The stock solution was treated with ultrasonic waves until the test item had completely dissolved. The 100-fold dilution in DMEM glutamax of 200, 100, 50, 25, 13, 6.3 and 3.1 mM showed moderate precipitate and were therefore not suitable to test. The 100-fold dilution of the 1.6 mM DMSO stock showed slight precipitation. This concentration was selected as highest concentration for the main assay (limit of solubility).
- Preparation of the test chemical serial dilutions
In the main experiments the test item was dissolved in DMSO at 1.6 mM (clear colourless
solution). From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series).
The stock and spike solutions were diluted 25-fold with exposure medium. These solutions
were diluted 4-fold with exposure medium in the assay resulting in final test concentrations of
16, 7.8, 3.9, 2.0, 0.98, 0.49, 0.24, 0.12, 0.061, 0.031, 0.015 and 0.008 μM (final concentration
DMSO of 1%).
- Preparation of the positive controls
The positive control used in the case of KeratinoSensTM is Ethylene glycol dimethacrylate
(Sigma, Zwijndrecht, The Netherlands), for which a 2-fold dilution series ranging from 0.78
to 25 mM were prepared in DMSO and diluted as described in paragraph 4.4.1, so that the
final concentration of the positive control ranges from 7.8 to 250 μM (final concentration
DMSO of 1%).
- Preparation of the solvent, vehicle and negative controls
The vehicle control was 1% DMSO in exposure medium. Eighteen wells were tested per plate.
On each plate three blank wells were tested (no cells and no treatment).

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 3
- Number of repetitions: 2
- Test chemical concentrations
- Application procedure
- Exposure time: 48 hours
- Study evaluation and decision criteria used
A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in
2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered
negative:
1. The Imax is equal or higher than (≥) 1.5-fold and statistically significantly different as
compared to the vehicle (negative) control (as determined by a two-tailed, unpaired
Student’s t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction
of luciferase activity ≥ 1.5-fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 μM (or < 200 μg/mL for test chemicals with no
defined MW)
4. There is an apparent overall dose-response for luciferase induction
Negative results obtained with concentrations <1000 μM or 200 μg/mL and which do not
reach cytotoxicity (< 70% viability) at the maximal tested concentration should be considered
as inconclusive.
- Description on study acceptance criteria
a) The luciferase activity induction obtained with the positive control, Ethylene glycol
dimethacrylate, should be statistically significant equal or above the threshold of 1.5
in at least one of the tested concentrations (from 7.8 to 250 μM).
b) The EC1.5 of the positive control should be within two standard deviations of the
historical mean. Moreover, the induction for Ethylene glycol dimethacrylate at 250
μM should be higher than 2-fold. If the latter criterion is not fulfilled, the doseresponse
of Ethylene glycol dimethacrylate should be carefully checked, and tests may
be accepted only if there is a clear dose-response with increasing luciferase activity
induction at increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the
vehicle (negative) control DMSO should be below 20% in each repetition which
consists of 18 wells tested. If the variability is higher, results should be discarded.
If the variability is higher, a maximum of three of the eighteen wells may be excluded
based on the Dixon’s Q-test. If the variability is still higher, the results should be
discarded.

LUCIFERASE ACTIVITY MEASUREMENTS
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate
(lyophilized) from Promega (Leiden, The Netherlands) were mixed together. The assay plates
were removed from the incubator and the medium is removed. Then 200 μL of the Steady-
Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was
added to each well. The plates were shaken for at least 5 minutes at room temperature. Plates
with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess
the quantity of luciferase (integration time two seconds).

DATA EVALUATION
- Cytotoxicity assessment
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure
time with fresh DMEM Glutamax containing MTT and cells were incubated for 3 - 4 hours at 37°C ± 1.0°C in the presence of 5% CO2. The MTT medium was then removed and cells were
lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

EGDMA (120 M) [442D]

Positive control results:

Experiment 1
The positive control Ethylene glycol dimethacrylate caused a dose related induction of
the luciferase activity. The Imax was 3.58 and the EC1.5 42 μM.
Experiment 2
The positive control Ethylene glycol dimethacrylate caused a dose related induction of
the luciferase activity. The Imax was 3.57 and the EC1.5 34 μM.

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

1.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

1.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

data inconclusive

Other effects / acceptance of results:

Experiment 1
• No precipitation was observed at the start and end of the incubation period in the 96-well
plates.
• The test item showed no toxicity. The viability of the cells was higher than 70% at all test
concentrations and therefore no IC30 and IC50 values could be calculated.
• No luminescence activity induction compared to the vehicle control was observed at any
of the test concentrations after treatment with the test item. The Imax was 1.30 and
therefore no EC1.5 could be calculated.

Experiment 2
• No precipitation was observed at the start and end of the incubation period in the 96-well
plates.
• The test item showed no toxicity. The viability of the cells was higher than 70% at all test
concentrations and therefore no IC30 and IC50 values could be calculated.
• A non-dose related luminescence activity induction was observed after treatment with the
test item. The Imax was 1.60 and the EC1.5 0.82 μM (as result of a fluctuation). Since this
was a clear outlier compared to the other dose levels in this experiment and the results
observed in the first experiment, it is likely the fluctuation was caused by an incidental
fluctuation and therefore considered not biologically relevant.

Interpretation of results:

other: inconclusive

Conclusions:

The test item showed no toxicity (no IC30 and IC50 value) in both experiments. No
biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any
of the test concentrations in the first experiment. An induction of the luciferase activity (EC1.5
value of 0.82 μM) was measured in the second experiment as result of a fluctuation up to an
Imax of 1.60-fold. Since this was a clear outlier compared to the other dose levels in this
experiment and the results observed in the first experiment, it is likely the fluctuation was
caused by an incidental fluctuation and therefore considered not biologically relevant. The
maximum luciferase activity induction (Imax) was 1.30-fold and 1.60-fold in experiment 1 and
2 respectively. The test item is classified as inconclusive in the KeratinoSensTM assay since
negative results (<1.5-fold induction; excluding the outlier value in the second experiment)
were observed at test concentrations < 1000 μM with a cell viability of >70% compared to the
vehicle control.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 22 to 24 Feb 2021

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

Batch No.: 465884
Purity: 100%

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions
A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10.0 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10.2 mg of SPCL in 19.69 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.
- Preparation of the test chemical solutions
750 μL Stock solution of 0.667 mM SPCL + 250 μL 211996/A test solution (100 mM)
- Preparation of the positive controls, reference controls and co-elution controls
Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were
prepared in amber vials by mixing 750 μL of the 0.667 mM SPCC stock solution with 250 μL
ACN.
Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were
prepared in amber vials by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL
ACN.

INCUBATION
- Incubation conditions: 25±2.5°C
- Precipitation noted: Prior to HPLC analysis the samples were visually inspected for precipitation. The test item samples that showed precipitation or phase separation were centrifuged (at 400 g) for 5 minutes at room temperature and supernatant was transferred to a new vial.

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys: 1.0
- Verification of the suitability of the HPLC for test chemical and control substances

DATA EVALUATION
- Cys and Lys peptide detection wavelength: 220 nm. The ratio of the 220 nm peak area and the
258 nm peak was used as an indicator of co-elution.

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Group:

test chemical

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

0.7 %

At concentration:

100 mM

Negative controls validity:

valid

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

1.9 %

At concentration:

100 mM

Negative controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

In the cysteine reactivity assay the test item showed 0.7% SPCC depletion while in the lysine reactivity assay the test item showed 1.9% SPCL depletion. The mean of the SPCC and SPCL depletion was 1.3% and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Interpretation of results:

GHS criteria not met

Conclusions:

The mean of the SPCC and SPCL depletion was 1.3% and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin sensitisation in vitro:

DPRA, OECD 442C: negative;

KeratinoSens, OECD 442D: inconclusive

Based on criteria in Table 3.4.2 of Regulation (EC) 1272/2008 and amendments, classification cannot be concluded, further studies need to be performed to assess the skin sensitisation of the substance.