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Description of key information

Study conducted to recognised testing guidelines with GLP certification

Key value for chemical safety assessment

Skin sensitisation

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Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 March 2018 to 14 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
February, 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
Valid in-vitro test rather than in-vivo test.
Specific details on test material used for the study:
Appearance: Colourless to pale yellow liquid
Purity/Composition: 100% (UVCB)
Test item storage: At room temperature

Details on study design:
The study was conducted to investigate the potential of the test material to induce genes that are regulated by the antioxidant response element (ARE). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
The ARE-Nrf2 luciferase test method utilises an immortalised adherent cell line derived from HaCaT human keratinocytes. The cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 promoter fused with the ARE from a gene known to be up-regulated by contact sensitisers.
The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by
luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic substances.

Specifications:
KeratinoSens™ cell line supplied by Givaudan Schweiz, Duebendorf, Switzerland.

Identification:
The test system was appropriately labelled with the study number, assay type, experiment number and test/positive/negative control.

Preparation of Cultures:
A fresh vial of cells was used for each experimental occasion and cultured using Dulbecco’s modified Eagle medium (DMEM) containing serum and Geneticin.

Treatment Plate Preparation:
The cells were 80-90% confluent (see Section 9 for details of protocol deviations). On the day prior to treatment, cells were harvested and distributed into 96-well plates (10000 cells/well) and incubated at 37±1°C, 5% (v/v) CO2, for 24±1 hours.
For each repetition, three replicates were used for the luciferase activity measurements and one parallel replicate used for the cell viability assay.

Treatment:
At the end of the 24-hour incubation period, the medium was removed and replaced with fresh culture medium (containing serum but without Geneticin) to which test article and control formulations were added.
One well per plate was left empty (no cells and no treatment) to assess background values.
Each plate was sealed and incubated at 37±1°C, 5% (v/v) CO2 in air, in a humidified environment for 48±1 hours.
For each test article and positive control, one experiment was needed to derive a prediction (positive or negative), consisting of at two independent repetitions each containing three replicates of each concentration.
The data for repetition 1 was obtained from a repeat experiment as the initial experiment did not meet the acceptance criteria for the positive or negative controls.
The data from the initial experiment has not been reported.
Discordant results were obtained between the two repetitions, therefore a third repetition containing three replicates was performed.
Each independent repetition was performed on a different day with fresh stock solutions of chemicals and independently harvested cells. The cells came from different passages.

Cytotoxicity Assessment:
After the 48-hour exposure period, the medium was replaced with fresh medium containing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).The plate was sealed and incubated for 4 hours at 37±1°C, 5% (v/v) CO2.
The MTT medium was removed and SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator at 37±1°C, 5% (v/v) CO2 in air and left overnight.
After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm using a SpectraMax M2e.

Luciferase Activity Measurements;
After the 48-hour exposure period, the cells were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plates were then incubated for 20 minutes at 25±2°C, loaded into the luminescence plate
reader and read using the following parameters: 100 µL injection (Luciferase assay substrate), 15 second delay, 7 second luminescence integration time.
Positive control results:
The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.27 and the EC1.5 85 µM.
The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.73 and the EC1.5 68 µM.
Key result
Parameter:
other: EC1.5
Remarks:
µg/mL
Run / experiment:
Experiment 1
Value:
0.66
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: EC1.5
Remarks:
µg/mL
Run / experiment:
Experiment 2
Value:
1.2
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: Imax
Run / experiment:
Experiment 1
Value:
3.52
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: Imax
Run / experiment:
Experiment 2
Value:
3.71
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: IC30
Remarks:
µg/mL
Run / experiment:
Experiment 1
Value:
4.3
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: IC30
Remarks:
µg/mL
Run / experiment:
Experiment 2
Value:
4.3
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: IC50
Remarks:
µg/mL
Run / experiment:
Experiment 1
Value:
4.9
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: IC50
Remarks:
µg/mL
Run / experiment:
Experiment 2
Value:
4.8
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation

Photomer 4250 was evaluated for the ability to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway.  An overview of the viability and luciferase activity induction is summarized in Table 1 and Figure 2-3.  The results of the positive control are summarized in Table 2 and Figure 4-5.  An overview of EC1.5, Imax, IC30 and IC50 values is given in Table 3.  The individual raw data are presented in Appendix 3 and Appendix 4.  The historical control data are presented in Appendix 5.

Two independent experiments were performed.  The cells were in these experiments incubated with Photomer 4250 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.

Experiment 1

•       Precipitate was observed at the start of the incubation period at the top dose level of 400 µg/mL and no precipitation was observed at the end of the incubation period.

•       Photomer 4250 showed toxicity.  The calculated IC30 was 4.3 1.2 and the calculated IC50 was 4.9 µg/mL.

•       A dose related luminescence activity induction was observed after treatment with Photomer 4250.  The Imax was 3.52 and the EC1.5 0.66 µg/mL.

•       The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity.  The Imax was 2.27 and the EC1.5 85 µM.

Experiment 2

•       Precipitate was observed at the start of the incubation period at the top dose level of 400 µg/mL and no precipitation was observed at the end of the incubation period.

•       Photomer 4250 showed toxicity.  The calculated IC30 was 4.3 µg/mL and the calculated IC50 was 4.8 µg/mL.

•       A dose related luminescence activity induction was observed after treatment with Photomer 4250.  The Imax was 3.71 and the EC1.5 1.2 µg/mL.

•       The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity.  The Imax was 2.73 and the EC1.5 68 µM.  

Both tests passed the acceptance criteria:

•       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 (79 µM and 68 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 2.73-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% (11% and 14% in experiment 1 and 2, respectively).

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

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In conclusion, Photomer 4250 is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate the ability of Photomer 4250 to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSensTM assay.

The study procedures described in this report were based on the most recent OECD guideline.

The batch of Photomer 4250 was a colourless to pale yellow liquid.  Photomer 4250 was dissolved in dimethyl sulfoxide at 40 mg/mL.  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 0.20 – 400 mg/mL (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 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 (85 µM and 68 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 2.73-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% (11% and 14% in experiment 1 and 2, respectively).

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

 

Photomer 4250 showed toxicity (IC30 values of 4.3 µM and 4.3 µM and IC50 values of 4.9 µM and 4.8 µM in experiment 1 and 2, respectively).  A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 0.66 µM and 1.2 µM in experiment 1 and 2, respectively) was measured in both experiments.  The maximum luciferase activity induction (Imax) was 3.52-fold and 3.71-fold in experiment 1 and 2 respectively.  Photomer 4250 is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed at test concentrations < 200 µg/mL with a cell viability of >70% compared to the vehicle control.

In conclusion, Photomer 4250 is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Endpoint:
skin sensitisation: in chemico
Type of information:
calculation (if not (Q)SAR)
Adequacy of study:
supporting study
Study period:
March 2018
Reliability:
1 (reliable without restriction)
Justification for type of information:
DEREK NEXUS is a knowledge-based system that contains 90 alerts for skin sensitization
based on the presence of molecular substructures.
Qualifier:
no guideline required
Principles of method if other than guideline:
DEREK NEXUS is a knowledge-based system that contains 90 alerts for skin sensitization
based on the presence of molecular substructures. LHASA (see Appendix I) has inserted
validation comments for the skin sensitization alerts.



The level of likelihood of a structure being sensitizing to skin is expressed in terms of:
Certain There is proof that the proposition is true.
Probable There is at least one strong argument that the proposition is true and there are no arguments against it.
Plausible The weight of evidence supports the proposition.
Equivocal There is an equal weight of evidence for and against the proposition.



The default of DEREK NEXUS for the level of likelihood, mentioning all alerts which are evaluated as being equivocal or greater was used in this assessment.

If a substance is predicted to be no skin sensitizer, DEREK NEXUS contains an expert- derived functionality to provide negative predictions for skin sensitization. This functionality further evaluates those compounds which do not fire any skin sensitization alerts in DEREK NEXUS. The query compound is compared to a Lhasa reference set of Ames test or skin sensitization data, producing the following outcomes:
• In compounds where all features in the molecule are found in accurately classified compounds from the reference set, a negative prediction is displayed: inactive.
• For those query compounds where features in the molecule are found in non-alerting skin sensitizers in the Lhasa reference set, the prediction remains negative and the misclassified [1] features are highlighted to enable the negative prediction to be verified by expert assessment.
• In cases where features in the molecule are not found in the Lhasa reference set, the prediction remains negative and the unclassified [2] features are highlighted to enable the negative prediction to be verified by expert assessment.


If a substance is predicted to be a skin sensitizer, its potency is predicted by DEREK NEXUS by calculating an EC3 value based on experimental data from the closest structurally-related substances (at least 3 substances should be present) using the following equation:

EC3Q = MWQ /(Σ ωNN / Σ TNN)

MW = molecular weight
T = Tanimoto similarity score
ω = weighting factor = (MWNN/EC3) * TNN
Q = query compound
NN = nearest neighbour

The EC3 is the estimated concentration needed to produce a stimulation index of 3.

[1] Misclassified features are those that have been derived from non-alerting mutagens in the Lhasa Ames test
reference set
[2] Unclassified features are those that have not been found in the Lhasa Ames test reference set
GLP compliance:
no
Justification for non-LLNA method:
Supporting data for non-animal test
Specific details on test material used for the study:
Identification: Photomer 4250
Chemical name: 1H-Azepine-1-propanoic acid, hexahydro-, 2,2-bis[[(1-oxo-2-propen-1-yl)oxy]methyl]butyl ester
CAS Number: 73003-78-8
Molecular weight: 395.49
Molecular formula: C21H33NO6


Parameter:
other: skin sensitisation
Run / experiment:
modelling data
Remarks on result:
positive indication of skin sensitisation
Conclusions:
DEREK NEXUS version 6.0.1 yielded an alert for Photomer 4250 for skin sensitization based on the presence of the alpha,beta-unsaturated ester groups. Photomer 4250 is predicted to be sensitizing to the skin (plausible). DEREK NEXUS predicts an EC3 of 0.74% (strong sensitizer) for the alpha,beta-unsaturated ester groups based on data on closest structurally-related substances. The mechanism for the alert is thought to occur through Michael addition.

Executive summary:

The objective of this study was to obtain a prediction on the potential for skin sensitization of the test item 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 yielded an alert for Photomer 4250 for skin sensitization based on the presence of the alpha,beta-unsaturated ester groups. Photomer 4250 is predicted to be sensitizing to the skin (plausible). DEREK NEXUS predicts an EC3 of 0.74% (strong sensitizer) for the alpha,beta-unsaturated ester groups based on data on closest structurally-related substances. The mechanism for the alert is thought to occur through Michael addition.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Justification for classification or non-classification

Photomer 4250 is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) and therefore classified as skin sensitising.