[No public or meaningful name is available]

Endpoint summary

Description of key information

In a direct peptide reactivity assay (DPRA) according to OECD guideline 442C, the test item was positive and was classified in the “moderate reactivity class” when using the Cysteine 1:10 prediction model. In the ARE-Nrf2 Luciferase Test according to OECD guideline 442D, the test item is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in the report.

 

Conclusion: The test item was positive in the DPRA and ARE-Nrf2 Luciferase Test Method. According to the vitro/in chemico testing strategy (OECD guidelines 442C-E), the test item is classified as sensitising, if two of the three key events of the skin sensitisation AOP are found to be positive. Thus, it is concluded that the test item is a sensitiser. 

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:

weight of evidence

Study period:

2019-03-13 to 2019-05-27

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)

Version / remarks:

Adopted 06-2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Justification for non-LLNA method:

The KeratinoSens test (ARE-Nrf2 luciferase reporter assay) is part of an in vitro/in chemico testing strategy (OECD guidelines 442C-E) addressing The second Adverse Outcome Pathway Key Event of inflammatory responses as well as gene expression associated with specific cell signaling (stress) pathways such as the antioxidant/ electrophile response element (ARE)-dependent pathways. Mechanistically-based in chemico and in vitro test methods addressing the first three key events of the skin sensitisation AOP have been adopted for contributing to the evaluation of the skin sensitisation hazard potential of chemicals.

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
- Preparation of the test chemical serial dilutions: From the 200 mM stock solution 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 controls: Ethylene dimethacrylate glycol was used as positive control. A 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted in exposure medium so that the final concentration of the positive control ranged from 7.8 to 250 μM (final concentration DMSO of 1%).
- Preparation of the vehicle control: The vehicle control was 1% DMSO in exposure medium.
- Stable dispersion obtained: Yes, no precipitation occured.

DOSE RANGE FINDING ASSAY:
- Solubility in solvents: The test item was dissolved in DMSO to a final concentration of 200 mM (clear colorless solution).
- Solubility in incubation medium: The 100-fold dilution of the 200 mM DMSO stock in DMEM glutamax formed a homogeneous solution (no precipitation).
- Cytotoxicity assessment performed: No.
- Final concentration range selected on basis of: 2000 µM were selected as highest concentration for the main assay based on the highest dose required in the current guideline.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 3
- Number of repetitions: 3
- Test chemical concentrations: 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM
- Application procedure: 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.
- Exposure time: 48 hours ± 1
- Study evaluation and decision criteria used: See "Any other information on material and methods incl. tables" and "Attached background material"
- Description on study acceptance criteria: See "Acceptance criteria" in "Any other information on material and methods incl. tables"

SEEDING AND INCUBATION
- Seeding conditions: 10,000 cells/well, passage 6 (experiment 1), passage 8 (experiment 2), passage 10 (experiment 3)
- Incubation conditions: At 37±1.0°C in the presence of 5% CO2
- Precipitation noted: No

LUCIFERASE ACTIVITY MEASUREMENTS
- Choice of luminometer: TECAN Infinite® M200 Pro Plate Reader to (integration time two seconds).
- Plate used: 96-well plates, manufacturer or wells type not indicated
- Lysate preparation: By addition of Steady-Glo Luciferase substrate solution (prior to addition, it was mixed 1:1 with exposure medium)

DATA EVALUATION
- 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 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.
- Prediction model used: See "Data interpretation" in "Any other information on material and methods incl. tables".

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

EGDMA (120 M) [442D]

Positive control results:

The fold luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was 2.49 (experiment 1), 3.24 (experiment 2) and 2.49 (experiment 3)

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

Imax [442D]

Value:

947 %

Cell viability:

No cytotoxicity was observed (no IC30 and IC50 value)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

128 %

Cell viability:

No cytotoxicity was observed (no IC30 and IC50 value)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

723 %

Cell viability:

No cytotoxicity was observed (no IC30 and IC50 value)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

EC 1.5 [442D]

Value:

13 µM

Cell viability:

No cytotoxicity was observed (no IC30 and IC50 value)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Cell viability:

No cytotoxicity was observed (no IC30 and IC50 value)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

21 µM

Cell viability:

No cytotoxicity was observed (no IC30 and IC50 value)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Not indicated

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.

Interpretation of results:

other: Activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes

Conclusions:

In conclusion, the test item 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 GLP compliant study according to OECD guideline 442D was to evaluate the ability of the test item to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay. The study procedures described in this report were based on the most recent OECD guideline. 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. Three independent experiments were performed.

All 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 (41, 31 and 50 μM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.49-fold, 3.24-fold and 2.49-fold in experiment 1, 2 and 3, respectively).


• Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (12%, 12% and 9.6% in experiment 1, 2 and 3, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly. In the first experiment, the test item showed no toxicity (no IC30 and IC50 value). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 value of 21 μM) was measured. The maximum luciferase activity induction (Imax) was 7.23-fold leading to an individual run conclusion of positive.

In the second experiment, 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. The maximum luciferase activity induction (Imax) was 1.28-fold leading to an individual run conclusion of negative.

Since the first two experiments were not concordant a third experiment was performed to provide a final conclusion.

In the third experiment, the test item showed no toxicity (no IC30 and IC50 value). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 value of 13 μM) was measured. The maximum luciferase activity induction (Imax) was 9.47-fold leading to an individual run conclusion of positive.

The test item is classified as positive in the KeratinoSens assay since positive results (>1.5-fold induction) were observed at test concentrations < 1000 μM with a cell viability of >70% compared to the vehicle control in two out of three experiments. In conclusion, the test item is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019-03-19 to 2019-05-03

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)

Version / remarks:

2015-02-04

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

The DPRA assay is part of an in vitro/in chemico testing strategy (OECD guidelines 442C-E) addressing The Adverse Outcome Pathway Key Event of covalent binding to proteins. Mechanistically-based in chemico and in vitro test methods addressing the first three key events of the skin sensitisation AOP have been adopted for contributing to the evaluation of the skin sensitisation hazard potential of chemicals.

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:
Cysteine: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 11.6 mg of cysteine peptides in 23.15 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
Lysine: A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10.0 mg of lysine peptides in 19.31 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.
- Preparation of the test chemical solutions: No correction for the purity/composition of the test item was performed. Solubility of the test item in acetonitrile was assessed before performing the DPRA. It dissolved the test item completely, i.e. by visual inspection the solution was not cloudy nor had noticeable precipitate. Test item stock solutions were prepared freshly for each reactivity assay. For both the cysteine and lysine reactivity assay, 33.57 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1803 μL ACN after vortex mixing to obtain a 100 mM solution. 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.

- Preparation of the positive controls, reference controls and co-elution controls:
Positive controls: The cinnamic aldehyde positive control samples (PC) were prepared with 750 μL Stock solution of 0.667 mM cysteine/lysine peptides and 250 μL Cinnamic aldehyde solution (100 mM in ACN)
Reference controls: Three 0.5 mM peptide reference control (RC) solutions (RCA, RCB and RCC) were prepared in amber vials by mixing 750 μL of the 0.667 mM cysteine/lysine peptide stock solution with 250 μL ACN.
Co-elution controls: The co-elution control (CC) samples were prepared with 750 μL Ammonium acetate buffer pH 10.2 and 250 μL test item solution (100 mM)

INCUBATION
- Incubation conditions: 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 24.2 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.
- Precipitation noted: Prior to HPLC analysis the samples were visually inspected for precipitation. No precipitate or phase separation was observed in any of the samples.

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys: See "Any other information on material and mehtods incl. table".
- Verification of the suitability of the HPLC for test chemical and control substances: See "Any other information on material and mehtods incl. table".

DATA EVALUATION
- Cys and Lys peptide detection wavelength: A220/A258

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Positive control results:

The acceptance criteria were met for the positive control with a cysteine peptide depletion value of 73.8% and a mean lysine peptide depletion value of 65.0%.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean lysine depletion

Remarks on result:

not determinable

Remarks:

A baseline interference was observed at the retention time of the lysine peptide. Since an accurate integration of the lysine peptide signal was not possible, the percent lysine depletion was not calculated and the cysteine 1:10 prediction model was used for reactivity classification.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

84.8 %

At concentration:

0.5 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Cysteine 1:10 prediction model

Outcome of the prediction model:

moderate reactivity [in chemico]

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, see "Attached background material"
- Acceptance criteria met for positive control: Yes, see "Attached background material"
- Acceptance criteria met for reference controls A to C: Yes, see "Attached background material"
- Acceptance criteria met for co-elution controls (Lysine and Cysteine): Yes, see "Attached background material"
- Acceptance criteria met for variability between replicate measurements: Yes, see "Attached background material"

Acceptability of the Cysteine Reactivity Assay

The correlation coefficient (r2) of the cysteine peptide standard calibration curve was 0.9998. Since the r2 was >0.99, the SPCC standard calibration curve was accepted. The mean peptide concentration of Reference Controls A was 0.511 ± 0.014 mM while the mean peptide concentration of Reference Controls C was 0.500 ± 0.004 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent cysteine peptide Depletion. The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 2.4%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time. The mean area ratio (A220/A258) of the Reference Control samples was 37.57. The mean A220/A258 ratio ± 10% range was 33.82-41.33. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred. The percent cysteine peptide depletion was calculated versus the mean cysteine peptide peak area of Reference Controls C. The mean percent cysteine peptide depletion for the positive control cinnamic aldehyde was 73.8% ± 0.2%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

 

Acceptability of the Lysine Reactivity Assay

The correlation coefficient (r2) of the lysine peptide standard calibration curve was 1.000. Since the r2 was >0.99, the SPCL standard calibration curve was accepted. The mean peptide concentration of Reference Controls A was 0.499 ± 0.008 mM while the mean peptide concentration of Reference Controls C was 0.500 ± 0.005 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent lysine peptide depletion.The CV of the peptide areas for the nine Reference Controls B and C was 2.6%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time. The mean area ratio (A220/A258) of the Reference Control samples was 31.62. The mean A220/A258 ratio ± 10% range was 28.46-34.78. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred. The Percent lysine peptide depletion was calculated versus the mean lysine peptide peak area of Reference Controls C. The mean Percent lysine peptide depletion for the positive control cinnamic aldehyde was 65.0% ± 0.3%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Interpretation of results:

other: Peptide reactivity

Conclusions:

In conclusion, this DPRA test is valid. The test item was positive in the DPRA and was
classified in the “moderate reactivity class” when using the Cysteine 1:10 prediction model.

Executive summary:

The objective of this GLP compliant study according to OECD guideline 442C was to determine the reactivity of the test item towards model synthetic peptides containing either cysteine or lysine. After incubation of the test item with either cysteine or lysine peptides, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and spectrophotometric detection at 220 nm and 258 nm. Cysteine or lysine peptides 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. The study procedures described in this report were based on the most recent OECD guideline. 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. An overview of the obtained assay validation parameters is presented in the table below.

Acceptability of the Direct Peptide Reactivity Assay (DPRA)

 

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPCL
Correlation coefficient (r^2) standard calibration curve	>0.99	0.9998	>0.99	1.000
Mean peptide concentration RC­A samples (mM)	0.50 ± 0.05	0.511 ± 0.014	0.50 ± 0.05	0.499 ± 0.008
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.500 ± 0.004	0.50 ± 0.05	0.500 ± 0.005
CV (%) for RC samples B and C	<15.0	2.4	<15.0	2.6
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	73.8	40.2-69.0	65.0
SD of peptide depletion cinnamic aldehyde (%)	<14.9	0.2	<11.6	0.3
SD of peptide depletion for the test item (%)	<14.9	1.4	<11.6	Int.

 

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A and C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the cysteine depletion for the test item, were all within the acceptability criteria for the DPRA. Upon preparation as well as after incubation of the cysteine or lysine 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 cysteine or lysine peptide depletion are presented in the table below. In the cysteine reactivity assay the test item showed 84.8% cysteine depletion while in the lysine reactivity assay the test item showed interference with cysteine. As a result the Cysteine 1:10 prediction model was used and the test item was considered to be positive in the DPRA and classified in the “moderate reactivity class”.

Endpoint conclusion

Additional information:

Skin Sensitisation in chemico (DPRA), WoE, RL1

The objective of this GLP compliant study according to OECD guideline 442C was to determine the reactivity of the test item towards model synthetic peptides containing either cysteine or lysine. After incubation of the test item with either cysteine or lysine peptides, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and spectrophotometric detection at 220 nm and 258 nm. Cysteine or lysine peptides 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. The study procedures described in this report were based on the most recent OECD guideline. 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. An overview of the obtained assay validation parameters is presented in the table below.

Acceptability of the Direct Peptide Reactivity Assay (DPRA)

 

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPCL
Correlation coefficient (r^2) standard calibration curve	>0.99	0.9998	>0.99	1.000
Mean peptide concentration RC­A samples (mM)	0.50 ± 0.05	0.511 ± 0.014	0.50 ± 0.05	0.499 ± 0.008
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.500 ± 0.004	0.50 ± 0.05	0.500 ± 0.005
CV (%) for RC samples B and C	<15.0	2.4	<15.0	2.6
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	73.8	40.2-69.0	65.0
SD of peptide depletion cinnamic aldehyde (%)	<14.9	0.2	<11.6	0.3
SD of peptide depletion for the test item (%)	<14.9	1.4	<11.6	Int.

 

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A and C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the cysteine depletion for the test item, were all within the acceptability criteria for the DPRA. Upon preparation as well as after incubation of the cysteine or lysine 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 cysteine or lysine peptide depletion are presented in the table below. In the cysteine reactivity assay the test item showed 84.8% cysteine depletion while in the lysine reactivity assay the test item showed interference with cysteine. As a result the Cysteine 1:10 prediction model was used and the test item was considered to be positive in the DPRA and classified in the “moderate reactivity class”.

 

 

Skin sensitisation in vitro (ARE-Nrf2 Luciferase Test Method), WoE, RL1

The objective of this GLP compliant study according to OECD guideline 442D was to evaluate the ability of the test item to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay. The study procedures described in this report were based on the most recent OECD guideline. 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. Three independent experiments were performed.

 

All 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 (41, 31 and 50 μM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.49-fold, 3.24-fold and 2.49-fold in experiment 1, 2 and 3, respectively).


• Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (12%, 12% and 9.6% in experiment 1, 2 and 3, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly. In the first experiment, the test item showed no toxicity (no IC30 and IC50 value). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 value of 21 μM) was measured. The maximum luciferase activity induction (Imax) was 7.23-fold leading to an individual run conclusion of positive.

 

In the second experiment, 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. The maximum luciferase activity induction (Imax) was 1.28-fold leading to an individual run conclusion of negative.

 

Since the first two experiments were not concordant a third experiment was performed to provide a final conclusion.

 

In the third experiment, the test item showed no toxicity (no IC30 and IC50 value). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 value of 13 μM) was measured. The maximum luciferase activity induction (Imax) was 9.47-fold leading to an individual run conclusion of positive.

 

The test item is classified as positive in the KeratinoSens assay since positive results (>1.5-fold induction) were observed at test concentrations < 1000 μM with a cell viability of >70% compared to the vehicle control in two out of three experiments. In conclusion, the test item is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

 

Conclusion: The test item was positive in the DPRA and ARE-Nrf2 Luciferase Test Method. According to the vitro/in chemico testing strategy (OECD guidelines 442C-E), the test item is classified as sensitising, if two of the three key events of the skin sensitisation AOP are found to be positive. Thus, it is concluded that the test item is a sensitiser. 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available in chemico and in vitro study, it is concluded that the test item is a skin sensitiser and should be classified under EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No 1272/2008, as amended for seventeenth time in Regulation (EU) No 2021/849.