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Description of key information

Skin sensitisation:

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.

A DEREK assessment did not yield skin sensitization alerts for test item and predicted the substance to be not skin sensitizing. The test item did not react with cysteine and lysine containing peptides in the DPRA. The test item is therefore not expected to bind to endogenous skin proteins. The results of the KeratinoSensTM assay indicated some activation of keratinocytes when exposed to test item. However, in two out of three experiments significant activation was only observed at the highest test concentrations (>1000 µM) and therefore the overall outcome was considered negative.

Taken all results together it is concluded that test item 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
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
2018-10-31
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:
See attached justification
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Software tool(s) used including version: DEREK NEXUS version 6.0.1
- Model(s) used: DEREK NEXUS
- Model description: see field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification'
GLP compliance:
no
Key result
Remarks on result:
no indication of skin sensitisation

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization of the test item. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer.

The test item is predicted to be not sensitizing to the skin.

Interpretation of results:
other: not sensitizing
Conclusions:
The test item is predicted to be not sensitizing to the skin.
Executive summary:

The potential for skin sensitization of test item was predicted with the in silico model DEREK NEXUS.

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization of the test item. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer.

The test item is predicted to be not sensitizing to the skin.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2019-01-16 to 2019-03-08
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:
2018
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes
Specific details on test material used for the study:
Batch No.: A7503
Purity: 99.89%
Details of test system:
Keratinoses transgenic cell line [442D]
Details on the study design:
VEHICLE
-Vehicle: dimethyl sulfoxide (DMSO)
-Justification for choice of vehicle: A solubility test was performed. The test item was dissolved in DMSO to a final concentration of 200 mM (clear colorless solution).

Dose Formulation
-Preparation of Test Item Stock, Spiking and Working Solutions: In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at
200 mM (clear colorless 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%).
-Preparation of the Positive Control: The positive control used in the case of KeratinoSensTM is Ethylene dimethacrylate glycol, for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted, the final concentration of the positive control ranges from 7.8 to 250 μM (final concentration DMSO of 1%).
-Preparation of the Solvent Control: The solvent control was 1% DMSO in exposure medium.

Test System
-Test System: A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSensTM cell line).
- Rationale: 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).
- Source: Givaudan (Duebendorf, Switserland).

Experimental Design
-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+5 in experiment 1, P+7 in experiment 2 and P+9 in experiment 3.
-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 ± 1 h at 37±1.0°C in the presence of 5% CO2. Initially, the results of the first experiment were unreliable (large variation in the viability of the triplicates) and therefore this experiment was repeated. In total 3 valid experiments were performed.
- Luciferase Activity Measurement: The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega 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 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
Positive control:
EGDMA (120 M) [442D]
Positive control results:
The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.

The EC1.5 of the positive control was between 5 and 125 μM (37.9 μM and 40.2 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.94-fold and 2.28-fold in experiment 1 and 2, respectively).
Key result
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
EC 1.5 [442D]
Value:
86 µM
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The maximum luciferase activity induction (Imax) was 2.24-fold.
Key result
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
EC 1.5 [442D]
Value:
1 875 µM
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The maximum luciferase activity induction (Imax) was 1.52-fold.
Key result
Group:
test chemical
Run / experiment:
run/experiment 3
Parameter:
EC 1.5 [442D]
Value:
1 501 µM
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The maximum luciferase activity induction (Imax) was 1.59-fold.
Other effects / acceptance of results:
OTHER EFFECTS:
-Precipitation: No precipitation was observed at the start and end of the incubation period in the 96-well plates for Experiment 1, 2 and 3.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control and negative (solvent) control: yes
-The test conditions were adequate and that the test system functioned properly.

The test item showed no toxicity. No IC30 and IC50 value was measured at any of the test concentrations in all experiments. 

In the first experiment, a biologically relevant induction of the luciferase activity (EC1.5 value of 86 µM) was measured. The maximum luciferase activity induction (Imax) was 2.24-fold. The result of this individual experiment is positive.

In the second experiment, a biologically relevant induction of the luciferase activity (EC1.5 value of 1875 µM) was measured. The maximum luciferase activity induction (Imax) was 1.52-fold. Since the EC1.5 > 1000 µM the result of this individual experiment is negative.

Since the first two experiments were not concordant a third experiment was performed. 

In the third experiment, a biologically relevant induction of the luciferase activity (EC1.5 value of 1501 µM) was measured. The maximum luciferase activity induction (Imax) was 1.59-fold. Since the EC1.5 > 1000 µM the result of this individual experiment is negative.

The test itemis classified as negativesince positive results (>1.5-fold induction) were observed at test concentrations > 1000 µM in two out of three experiments.

Interpretation of results:
other: negative
Conclusions:
The test item is classified as negative (no biologically relevant activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.
Executive summary:

To evaluate the ability of test item to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSensTM assay according to OECD guideline 442D.

The test item was dissolved in dimethyl sulfoxide at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 µM (2-fold dilution series). The highest test concentration was thehighest dose required in the current guideline. No precipitate was observed at any dose level tested. Three independent experiments were performed.

All experiments passed the acceptance criteria,the test conditions were adequate and that the test system functioned properly.

The test item showed no toxicity. No IC30 and IC50 value was measured at any of the test concentrations in all experiments. 

In the first experiment, a biologically relevant induction of the luciferase activity (EC1.5 value of 86 µM) was measured. The maximum luciferase activity induction (Imax) was 2.24-fold. The result of this individual experiment is positive.

In the second experiment, a biologically relevant induction of the luciferase activity (EC1.5 value of 1875 µM) was measured. The maximum luciferase activity induction (Imax) was 1.52-fold. Since the EC1.5 > 1000 µM the result of this individual experiment is negative.

Since the first two experiments were not concordant a third experiment was performed. 

In the third experiment, a biologically relevant induction of the luciferase activity (EC1.5 value of 1501 µM) was measured. The maximum luciferase activity induction (Imax) was 1.59-fold. Since the EC1.5 > 1000 µM the result of this individual experiment is negative.

The test itemis classified as negativesince positive results (>1.5-fold induction) were observed at test concentrations > 1000 µM in two out of three experiments.

In conclusion, the test item is classified as negative (no biologically relevantactivation of theantioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2019-01-24 to 2019-01-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
other: Direct Peptide Reactivity Assay (DPRA)
Specific details on test material used for the study:
Batch No.: A7503
Purity: 99.89%
Details of test system:
cysteine peptide, (Ac-RFAACAA-COOH)
lysine peptide (Ac-RFAAKAACOOH)
Details on the study design:
Test system
- Test system: Synthetic peptides containing cysteine (SPCC) or synthetic peptides containing lysine (SPCL)
- Rationale: Recommended test system in the international OECD guideline for DPRA studies.
- Source: JPT Peptide Technologies GmbH

Experimental Design
- Test Item Preparation: 28.44 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1711 μL ACN after vortex mixing to obtain a 100 mM solution..
- Preparation of Solutions for Cysteine Reactivity Assay:
Synthetic Peptide Containing Cysteine (SPCC) Stock Solution: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving
12.2 mg of SPCC in 24.35 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
SPCC Reference Control Solutions: 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.
- Preparation of Solutions for Lysine Reactivity Assay:
Synthetic Peptide Containing Lysine (SPCL) Stock Solution: 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.
SPCL Reference Control Solutions: 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.
- Sample Incubations: 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 23.6 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.
- HPLC Analysis: SPCC and SPCL peak areas in the samples were measured by HPLC.
- Other measurement: Prior to HPLC analysis the samples were visually inspected for precipitation.
Vehicle / solvent:
acetonitrile
Positive control:
cinnamic aldehyde
Positive control results:
The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 71.0% ± 0.2%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).
The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 61.4% ± 1.3%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).
Key result
Group:
test chemical
Run / experiment:
mean
Parameter:
cysteine depletion
Value:
3.4 %
At concentration:
100 mM
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Group:
test chemical
Run / experiment:
mean
Parameter:
lysine depletion
Value:
0 %
At concentration:
100 mM
Vehicle controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- Precipitation: Cysteine Reactivity Assay: No precipitate or phase separation was observed in any of the samples; Lysine Reactivity Assay: No precipitate or phase separation was observed in any of the samples.
- Test Acceptability: all acceptability criteria were met this DPRA is considered to be valid.

In the cysteine reactivity assay the test item showed 3.4% SPCC depletion while in the lysine reactivity assay the test item showed 0.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 1.7% and as a result the test item was 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.

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 prediction model.
Executive summary:

The reactivity of test item towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was determined according to OECD guideline 442C.

After incubation of test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test chemical to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study.

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. An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table below. In the cysteine reactivity assay the test item showed 3.4% SPCC depletion while in the lysine reactivity assay the test item showed 0.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 1.7% 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.

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. test item was 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.

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:

Based on the in silico/in chemico/in vitro data available, the substance can be concluded to have no skin sensitizing properties.

The substance does not have to be classified according to Regulation 1272/2008 and amendments.