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Description of key information

Skin sensitisation:

A DEREK assessment, DPRA assay and a KeratinoSensTM assay were available.

A DEREK prediction on the skin sensitizing potential of the test item was negative for skin sensitization and predicted the substance to be a non-sensitizer.

The test item did not react with cysteine resulting in a negative outcome for the DPRA (co-elution was observed in the lysine reactivity assay and therefore binding to lysine could not be determined).

The KeratinoSensTM assay did not show a biologically relevant activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway when exposed to the test item and was therefore concluded to be negative.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
2018-11-20
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. In this assessment version 6.0.1 of DEREK NEXUS was used.

 

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 chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2019-2-11 to 2019-2-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed to current OECD guidelines with no significant deviations and run in OECD GLP certified lab.
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
Type of study:
other: Direct Peptide Reactivity Assay (DPRA)
Specific details on test material used for the study:
Purity: 99.7%
Batch No.: 80031745a
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: 28.50 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1582 µL ACN after vortex mixing to obtain a 100 mM solution.
- Preparation of Solutions for Cysteine Reactivity Assay
SPCC Stock Solution: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10.5 mg of SPCC in 20.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
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
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.
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 23.5 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-PDA analysis the samples were visually inspected for precipitation.
Positive control results:
The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 91.6% ± 0.1%. 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 47.9% ± 4.9%. 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
Run / experiment:
other: Cysteine Reactivity Assay
Parameter:
other: SPCC depletion%
Value:
0
Positive controls validity:
valid
Remarks on result:
other: the mean Percent SPCC Depletion for the test item
Key result
Run / experiment:
other: Lysine Reactivity Assay
Parameter:
other: SPCL depletion%
Positive controls validity:
valid
Remarks on result:
other: the test item co-eluted with SPCL and therefore the Percent SPCL Depletion could not be calculated.
Other effects / acceptance of results:
- Precipitation: No precipitate or phase separation was observed in any of the samples for both Cysteine Reactivity Assay and Lysine Reactivity Assay.
- Test Acceptability: all acceptability criteria were met this DPRA is considered to be valid.

In the cysteine reactivity assay the test item showed 0.0% SPCC depletion while in the lysine reactivity assay the test item showed interference with SPCL. As a result, the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivityclass” when using the Cysteine 1:10 prediction model.

Interpretation of results:
other: negative
Conclusions:
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 prediction model.
Executive summary:

The objective of this study was to determine the reactivity of the test substance towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) according to OCED Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)).

After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and spectrophotometric 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 item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

 

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.

In the cysteine reactivity assay the test item showed 0.0% SPCC depletion while in the lysine reactivity assay the test item showed interference with SPCL. 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 prediction model.

 

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. The test substance was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 prediction model.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2019-02-08 to 2019-03-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed to current OECD guidelines with no significant deviations and run in OECD GLP certified lab.
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 (incl. QA statement)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
The existing knowledge of the chemical and biological mechanisms associated with skin sensitization has been summarized in the form of an Adverse Outcome Pathway (AOP). The second key event in this AOP takes place in the keratinocytes and includes inflammatory responses as well as gene expression associated with specific cell signaling (stress) pathways such as the antioxidant/ electrophile response element (ARE)-dependent pathways.
The KeratinoSensTM test (an ARE-Nrf2 luciferase reporter assay) is proposed to address this second key event. Skin sensitizers have been reported to induce genes that are regulated by the antioxidant response element (ARE). Small electrophilic substances such as skin sensitisers can act on the sensor protein Keap1 (Kelch-like ECH-associated protein 1), by e.g. covalent modification of its cysteine residue, resulting in its dissociation from the transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2). The dissociated Nrf2 can then activate ARE-dependent genes such as those coding for phase II detoxifying enzymes. In the KeratinoSensTM cell line activation of the Nrf2 pathway results in luciferase expression which can be quantified easily.
Specific details on test material used for the study:
Purity: 99.7%
Batch No.: 80031745a
Details on the study 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+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 ± 1 h at 37±1.0oC in the presence of 5% CO2. Initially, experiment 1 did not pass all the acceptability criteria and therefore this experiment was repeated. 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)-2,5diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1; Sigma, Zwijndrecht, The Netherlands) 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 (Sigma, Zwijndrecht, The Netherlands) to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.
Positive control results:
Experiment 1:
The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.70 and the EC(1.5) 95 µM.

Experiment 2:
The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.43 and the EC(1.5) 85 µM.
Key result
Run / experiment:
other: 1
Parameter:
other: Imax
Remarks:
The maximal average fold induction of luciferase activity
Value:
0.95
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 2
Parameter:
other: Imax
Remarks:
The maximal average fold induction of luciferase activity value
Value:
1.43
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
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.
The EC1.5 of the positive control was within two standard deviations of the historical mean (95 µM and 85 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.70-fold and 2.43-fold in experiment 1 and 2, respectively).
Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (7.2% and 6.8% in experiment 1 and 2, respectively).

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 0.95 and therefore no EC1.5 could be calculated.  

The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity.  The Imax was 2.70 and the EC(1.5) 95 µM.  

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.  

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 1.43 and therefore no EC1.5 could be calculated.  

The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity.  The Imax was 2.43 and the EC(1.5) 85 µM.

Interpretation of results:
other: negative
Conclusions:
The test item 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 1.43-fold in experiment 1 and 2 respectively. The test item 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:

The objective of this study was to evaluate the ability of the test item to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay based on the most recent 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 the highest dose required in the current guideline. No precipitate was observed at any dose level tested. Two independent experiments were performed.

Both experiments passed the acceptance criteria:

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.  

The EC1.5 of the positive control was within two standard deviations of the historical mean (95 µM and 85 µM in experiment 1 and 2, respectively).  A dose response was observed and the induction at 250 µM was higher than 2-fold (2.70-fold and 2.43-fold in experiment 1 and 2, respectively).

Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (7.2% and 6.8% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.  

The test item 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 1.43-fold in experiment 1 and 2 respectively.  The test item is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 µM.

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

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

DEREK assessment:

The potential for skin sensitization of test item was predicted with the in silico model DEREK NEXUS. In this assessment version 6.0.1 of DEREK NEXUS was used.

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.

DPRA assay:

The objective of this study was to determine the reactivity of the test substance towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) according to OCED Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)).

After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and spectrophotometric 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 item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

 

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.

In the cysteine reactivity assay the test item showed 0.0% SPCC depletion while in the lysine reactivity assay the test item showed interference with SPCL. 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 prediction model.

 

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. The test substance was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 prediction model.

KeratinoSensTM assay:

The objective of this study was to evaluate the ability of the test item to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay based on the most recent 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 the highest dose required in the current guideline. No precipitate was observed at any dose level tested. Two independent experiments were performed.

Both experiments passed the acceptance criteria:

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.  

The EC1.5 of the positive control was within two standard deviations of the historical mean (95 µM and 85 µM in experiment 1 and 2, respectively).  A dose response was observed and the induction at 250 µM was higher than 2-fold (2.70-fold and 2.43-fold in experiment 1 and 2, respectively).

Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (7.2% and 6.8% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.  

The test item 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 1.43-fold in experiment 1 and 2 respectively.  The test item is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 µM.

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

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin sensitisation:

Negative result in DEREK assessment, DPRA assay and KeratinoSensTM assay.

According to Regulation (EC) No 1272/2008, table 3.4.2, this substance is not classified for this endpoint.