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Description of key information

A DEREK assessment, a DPRA and a KeratinoSensTM assay were performed in accordance with Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the strategy presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a.

Based on the results of these studies, using a weight of evidence approach, it is concluded that the substance is not a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Remarks:
In silico assessment
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
Not applicable
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
An in silico assesment is mentioned in the ECHA guidance as one of the non-animal data that may be provided as weight of evidence.
Principles of method if other than guideline:
An in silico assessment was performed using DEREK NEXUS (version 6.0.1).
GLP compliance:
no
Justification for non-LLNA method:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. An in silico assesment is mentioned in the ECHA guidance as one of the non-animal data that may be provided as weight of evidence.
Key result
Parameter:
other: Prediction on skin sensitization using DEREK NEXUS
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization. The substance is predicted to be a non-sensitizer.
Interpretation of results:
other: Study is part of a weight of evidence approach and is not used for classification on its own.
Conclusions:
DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization. The substance is predicted to be a non-sensitizer.
Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
26 Feb 2019 - 28 Feb 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The validated in chemico skin sensitization test is the DPRA assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as the in chemico test to be performed as part of weight of evidence.
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
4 February 2015
Deviations:
no
GLP compliance:
yes
Type of study:
direct peptide reactivity assay (DPRA)
Justification for non-LLNA method:
A validated in chemico skin sensitization test is the DPRA assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as one of the non-animal tests to be performed as part of weight of evidence.
Details on the study design:
TEST ITEM PREPARATION
Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. Acetonitrile (ACN) was evaluated and assessed to be suitable.
Test item stock solutions were prepared freshly for each reactivity assay.
For both the cysteine and lysine reactivity assay 34.53 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1504 µL ACN after vortex mixing to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.

TEST SYSTEM
Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL (JPT Peptide Technologies GmbH, Berlin, Germany). The peptides were stored in the freezer (≤ -15°C) for a maximum of 6 months.

Calibration curve SPCC and SPCL: according to guideline
Positive control: cinnamic aldehyde
Incubation: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25 ± 2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 24.8 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
Prior to HPLC analysis the samples were visually inspected for precipitation. None of the samples showed precipitation.

Analysis: All samples were analyzed according to the HPLC method presented in the below Table (See 'other information on materials and methods').
The HPLC sequences of the cysteine and lysine reactivity assay for the test item are presented in the other below Table (See 'other information on materials and methods').

DATA EVALUATION
The concentration of SPCC or SPCL was spectrophotometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards.
The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:
Percent Peptide Depletion= [1-(Peptide Peak Area in Replicate Injection (at 220 nm)/Mean Peptide Peak Area in Reference Controls (at 220 nm))]x100

In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample, a ratio in the range of 90% < mean area ratio of control samples < 110% gives a good indication that co-elution has not occurred.

DATA INTERPRETATION
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model (see table below), the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.
Key result
Run / experiment:
other: Cysteine Reactivity Assay
Parameter:
other: Mean SPCC depletion (%)
Value:
4.7
Negative controls validity:
valid
Remarks:
CV between reference controls: 0.4%
Positive controls validity:
valid
Remarks:
Mean percentage SPCC: 79.6 ± 0.0%
Remarks on result:
other: SD: 1.3%
Key result
Run / experiment:
other: Lysine Reactivity Assay
Parameter:
other: Mean SPCL depletion (%)
Value:
1
Negative controls validity:
valid
Remarks:
CV between reference controls: 0.7%
Positive controls validity:
valid
Remarks:
Mean percentage SPCL: 52.0 ± 2.5%
Remarks on result:
other: SD: 1.1%
Other effects / acceptance of results:
- Upon preparation as well as after incubation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples.
- No co-elution occured during both assays.

Assessment of the acceptability criteria (which were all met):

 

Cysteine reactivity assay

Lysine reactivity assay

Acceptability criteria

Results for SPCC

Acceptability criteria

Results for SPCL

Correlation coefficient (r2) standard calibration curve

>0.99

0.9999

>0.99

0.9999

Mean peptide concentration RC-A samples (mM)

0.50 ± 0.05

0.501 ± 0.001

0.50 ± 0.05

0.499 ± 0.001

Mean peptide concentration RC-C samples (mM)

0.50 ± 0.05

0.503 ± 0.001

0.50 ± 0.05

0.502 ± 0.006

CV (%) for RC samples

B and C

<15.0

0.4

<15.0

0.7

Mean peptide depletion cinnamic aldehyde (%)

60.8-100

79.6

40.2-69.0

52.0

SD of peptide depletion cinnamic aldehyde (%)

<14.9

0.0

<11.6

2.5

SD of peptide depletion for the test item (%)

<14.9

1.3

<11.6

1.1

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation.

Interpretation of results:
other: Study is part of a weight of evidence approach and is not used for classification on its own.
Conclusions:
The substance was tested negative in the DPRA and was classified in the "No or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.
Executive summary:

In an in chemico study, performed according to OECD guideline 442C and GLP principles, the reactivity of the substance towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was determined to assign the substance to one of four reactivity classes used to support the discrimination between skin sensitisers and non skin sensitisers. Following incubation of the test substance with either SPCC or SPCL, the relative peptide concentration was determined by HPLC with gradient elution and spectrophotometric detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in the prediction model. Acetonitrile was found to be an appropriate solvent to dissolve the test substance. Cinnamic aldehyde was used as a positive control. All acceptability criteria were met and therefore, the study was considered to be valid. Upon preparation as well as after incubation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples. No co-elution of the test item with SPCC or SPCL was observed. In the cysteine reactivity assay the test item showed 4.7% SPCC depletion while in the lysine reactivity assay the test item showed 1.0% SPCL depletion. The mean of the SPCC and SPCL depletion was therefore 2.9% 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:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
28 Feb 2019 - 22 Mar 2019
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:
25 June 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EURL ECVAM DB-ALM Protocol n° 155: KeratinoSens™
Version / remarks:
March 2018
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD 442D).
Details on the study design:
TEST ITEM PREPARATION
- A solubility test was performed. The test item was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 200 mM (light yellow solution). The 100-fold dilution of the 200 mM DMSO stock in DMEM glutamax formed also a homogeneous solution (no precipitation). This concentration was selected as highest concentration for the main assay (highest dose required in the current guideline; 2000 μM).
- In the main experiments the test item was dissolved in DMSO at 200 mM (light yellow solution). From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution 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 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 µM (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution.
Test item concentrations were used within 2.5 hours after preparation.

CONTROL ITEMS
- Positive control: ethylene dimethacrylate glycol (tested in triplicate)
Amount used: 0.78 to 25 mM in DMSO, diluted so that the final concentration ranged from 7.8 to 250 μM (final concentration DMSO of 1%)
- Negative control: eighteen wells per plate of a solvent control of 1% DSMO were tested
- Blank control: on each plate three blank wells were tested (no cells and no treatment)

TEST DESIGN
- Test system: The KeratinoSens™ cell line, having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used. Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.
- Plating of cells: For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+11 in experiment 1 and P+13 in experiment 2.
- Treatment of cells: The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours at 37±1.0 °C in the presence of 5% CO2. In total 2 valid experiments were performed.
- Luciferase activity measurement: 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).
- Cytotoxicity assessment: For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and subsequently cells were incubated for 3 - 4 hours at 37±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.

DATA ANALYSIS
According to OECD guideline.

DATA INTERPRETATION
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.

ACCEPTABILITY CRITERIA
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, 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 value of the positive control should be within two standard deviations of the historical mean (Mean: 58.5 µM; SD: 30.7 µM). Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol 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) 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.
Positive control results:
Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.27 and the EC1.5 105 µM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.08 and the EC1.5 111 µM.
Key result
Run / experiment:
other: Experiment 1
Parameter:
other: Imax
Value:
0.95
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: Experiment 2
Parameter:
other: Imax
Value:
1
Vehicle controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Both experiments passed the acceptance criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration (at 125 and at 250 µM, in both experiments).
b) The EC1.5 of the positive control was within two standard deviations of the historical mean (105 µM and 111 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.27-fold and 2.08-fold in experiment 1 and 2, respectively).
c) The average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (7.0% and 7.9% in experiment 1 and 2, respectively).

Precipitation:
In both experiments, no precipitation was observed at the start and end of the incubation period in the 96-well plates.

Cytotoxicity:
In both experiments, 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.

Luminescence activity induction:
In both experiments, no luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item (no EC1.5 value).
Interpretation of results:
other: Study is part of a weight of evidence approach and is not used for classification on its own.
Conclusions:
The substance is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 µM.
Executive summary:

A KeratinoSensTM assay was performed with the substance according to OECD 442D and GLP principles. Two independent experiments were performed. The cells were in these experiments incubated with the test item in a concentration range of 0.98 – 2000 µM (2-fold dilution steps) for 48 hours. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay. The experiments passed the acceptance criteria and no precipitation was observed at the start and end of the incubation period in the 96-well plates. The substance showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 0.95-fold and 1.00-fold in experiment 1 and 2 respectively. The substance is therefore classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 µM.

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

A DEREK prediction on the skin sensitizing potential of the substance was negative, the substance did not react with cysteine and lysine containing peptides resulting in a negative outcome for the DPRA and the KeratinoSensTM assay did not show a biologically relevant activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway when exposed to the substance and was therefore concluded to be negative.

Justification for classification or non-classification

Based on the results of the available studies, using a weight of evidence approach, the substance does not need to be classified for skin sensitization according to Regulation (EC) No. 1272/2008 (CLP Regulation).