2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-[(thiophen-2-yl)methyl]acetamide

Endpoint summary

Description of key information

- In chemico study: direct peptide binding assay (DPRA, similar to 442c, GLP, K, Rel.1): predicted to be a skin sensitiser

- In vitro study: activation of keratinocytes (KeratinoSens, similar to 442d, GLP, K, Rel.2): predicted to be a skin sensitiser

- HRIPT: not a sensitiser at 0.01% (n = 110)

- Repiratory sensitisation: no data available

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6-Sept-2013 to 07-Feb-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Non-animal testing is the default requirement for skin sensitisation.

Details on the study design:

The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item at the ratios 1:10 cysteine:test item and 1:50 lysine:test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with ultra-violet detection at 220 nm.
Peptide reactivity was reported as percent depletion based on the amount of peptide remaining in the sample relative to the average amount measured in the Quality Control samples.

Preparation of test item and controls:
- Vehicle: acetonitrile at 100 mM
- Positive control: Name : Cinnamaldehyde (CAS No. : 14371-10-9, Batch No. : STBC8461V, Supplier : Sigma-Aldrich)
- Co-elution control samples: In order to detect possible co-elution of the test item with a peptide, co-elution control samples were prepared on the day of the assay by incubating the test item formulation with each buffer used to dilute the peptides.
- Quality Control (QC) samples: In order to control the precision of the peptide quantification throughout the analytical sequence, QC samples were prepared on the day of the assay by diluting each peptide at 0.500 mM in acetonitrile.
- Test item and positive control preparation: Each chemical (test item or positive control) was pre-weighed and stored under appropriate conditions until ready to perform testing. The positive control was dissolved in acetonitrile at 100 mM. The test item was dissolved in the selected vehicle (acetonitrile) at 100 mM. This formulation had the aspect of a yellowish liquid. Each formulation was prepared within 1 hour. Two aliquots of 500 μL were sampled for chemical analysis and stored at -20°C until shipment or destruction. One of the two aliquots was then sent to the Sponsor on dry ice for chemical analysis at the Sponsor’s Test Site; the other aliquot was destroyed after receipt of the finalized analytical phase report.
- Chemical analysis of the dose formulations: The test item quantification in the dose formulation samples was performed at the Sponsor’s Test Site.The analytical method was developed in the Sponsor’s analytical facility (i.e. Test Site). This phase was not under GLP, indeed, this formulation analysis was performed in the spirit of, but not in full compliance with, the Good Laboratory Practice Regulations for Nonclinical Laboratory Studies; however, it was conducted in accordance with the applicable test site’s Standard Operating Procedures (SOPs) and the study protocol and was carried out to high scientific and facility quality standards.

Cysteine peptide:
. Peptide sequence : AC RFAACAA-COOH
. Peptide sequence synonyms : AC RFAACAA-OH; AC-RFAACAA-COOH or RFAACAA-COOH
The cysteine peptide solution was prepared in an aqueous phosphate buffer (pH 7.5) solution.

Lysine peptide
. Peptide sequence : AC-RFAAKAA-COOH
. Peptide sequence synonyms : AC RFAAKAA-OH; AC-RFAAKAA-COOH or RFAAKAA-COOH
The lysine peptide solution was prepared in an aqueous ammonium acetate buffer (pH 10.2) solution.

Study design:
- Preparation of the samples:
On the day of the assay, the following samples were prepared in triplicate except for the co-elution control samples for which only one sample was prepared per peptide.
- Co-elution control samples preparation
For the co-elution control with cysteine peptide: 50 μL of test item formulation was incubated with 750 μL of cysteine peptide dilution buffer (without cysteine peptide) and 200 μL of acetonitrile.
For the co-elution control with lysine peptide: In parallel, 250 μL of test item formulation was incubated with 750 μL of lysine peptide dilution buffer (without lysine peptide).
- Quality Control samples preparation
In a vial, acetonitrile was added to a volume of peptide solution (cysteine or lysine) to achieve a nominal concentration of 0.500 mM. The QC samples were prepared using different standard solutions to those used for the calibration curve.
- Cinnamaldehyde (positive control) depletion control samples preparation
For the reactivity of cinnamaldehyde with cysteine peptide: 50 μL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
For the reactivity of cinnamaldehyde with lysine peptide: In parallel, 250 μL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).
- Test item samples preparation
For the reactivity of test item with cysteine peptide: 50 μL of test item formulation was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
For the reactivity of test item with lysine peptide: In parallel, 250 μL of test item formulation was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).
- Incubation of the samples
All samples (co-elution control, QC, test item and positive control samples) were incubated during 24 (± 2) hours at room temperature and protected from light before injection onto the HPLC/UV system. At the end of the incubation period, a visual inspection of the samples was performed prior to HPLC analysis. No precipitate was observed in the vials incubated with the cysteine peptide, as a result, these vials were directly transferred onto the HPLC/UV system. However, some crystals were observed in the test item samples vials incubated with the lysine peptide and precipitates were observed in the positive control samples incubated with either cysteine or lysine peptides. Therefore these vials (test item samples incubated with cysteine and positive control samples incubated with lysine and cysteine) and QC sample vials as well, were centrifuged at 400 g for 5 minutes at room temperature. Since precipitates were still observed in the positive control samples incubated with the cysteine peptide, an additional centrifugation was performed on these vials at 2000 g for 10 minutes and at room temperature . Supernatants were then collected and were transferred onto the HPLC/UV system.
- Preparation of the calibration curve samples
One set of calibration standards was prepared with each analytical sequence by spiking both peptides (lysine and cysteine) in separate solutions of 20% acetonitrile:peptide dilution buffer to obtain at least six different concentration levels ranging from 0.045 to 0.900 mM. The calibration curves were defined by the relationships between the peak area of the peptide signal versus the nominal concentration. These curves were obtained by using the appropriate mathematical model.
- HPLC/UV analysis of the samples
The study samples were assayed in batches using HPLC/UV analysis. For each peptide, the analytical sequence included at least:
. one blank sample (peptide dilution buffer),
. one calibration curve (within the range defined during validation) injected at the beginning of the analytical batch,
. one co-elution control sample,
. three QC samples injected at least three times to bracket study samples,
. three cinnamaldehyde depletion control samples preceded and followed by one QC sample,
. three test item samples followed by QC samples.
The HPLC/UV method used for the samples analysis is described in CiToxLAB France internal analytical method. For the cysteine peptide, the whole analytical sequence was injected using 7 μL as the injection volume (as described in CiToxLAB France internal analytical method on DPRA). Since one of the acceptance criteria on QC samples was not reached (back-calculated concentrations out of ± 10% of the nominal concentration 0.500 mM), the obtained results were invalidated and the samples were re-injected on the HPLC/UV using a lower injection volume (5 μL) which is also described in CiToxLAB France internal analytical method on DPRA. Since all acceptance criteria then were met, the analytical sequence was considered as valid and only the results obtained with the injection volume of 5 μL were taken into account for the evaluation.

Positive control results:

Cinnamic aldehyde was used as a positive control.
- Result of positive control samples in cysteine assay: Mean % depletion = 100.0% and SD = 0.0%
- Result of positive control samples in lysine assay: Mean % depletion = 62.54% and SD = 0.8%
Mean depletion rate of Cinnamaldehyde: 81.27%
The mean percent peptide depletion values for the positive control with its standard deviation value were within the acceptability criteria for the DPRA assay (cysteine and lysine reactivity assays).

Key result

Parameter:

other: Mean % depletion in Lysine assay

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Value set to "0" as the mean % depletion value is <0

Key result

Parameter:

other: Mean % depletion in Cysteine assay

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Default value due to concentration below limit of quantification after depletion

Key result

Parameter:

other:

Remarks:

Mean depletion rate

Value:

50

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Highly reactive

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline: Not specified

See the attached document "Tables of results DPRA"

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Under the experimental conditions of this study, the test item was considered to be highly reactive with peptides and therefore has potential to cause skin sensitization.

Executive summary:

The skin sensitisation potential of the test item was evaluated using an in chemico direct peptide binding assay (DPRA) similarly to the OECD Guideline No. 442C and in compliance with GLP. The reactivity of the test item was evaluated by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item at the ratios 1:10 cysteine:test item and 1:50 lysine:test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with ultra-violet detection at 220 nm. Peptide reactivity was reported as percent depletion based on the amount of peptide remaining in the sample relative to the average amount measured in the Quality Control samples.

The acceptance criteria of the samples for the calibration curve, QC and positive control were satisfied. The study was therefore considered to be valid.

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute either with the lysine or the cysteine peptides. Mean percentage depletion values were calculated for each peptide:

. 100% for the cysteine peptide since the cysteine concentrations measured in each sample were below the limit of quantification after depletion (< 0.100 mM),

. for the lysine peptide, two out of three individual depletion values were found negative. The mean depletion value was therefore set to 0%.

The mean of the percentage cysteine and percentage lysine depletions was calculated to be 50.00%.

Accordingly, the test item was considered to be highly reactive.

Under the experimental conditions of this study, the test item was considered to be highly reactive with peptides and therefore has potential to cause skin sensitization.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 August 2013 - 24 Feb 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Historical acceptance range under development at the time of the test; Confluence of the cells for testing 60-90% instead of 80-90% in the guideline: lysis buffer 30 min instead of 20 min; OD lecture at 570nm instead of 600 nm.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

yes

Remarks:

Historical acceptance range under development at the time of the test; Confluence of the cells for testing 60-90% instead of 80-90% in the guideline: lysis buffer 30 min instead of 20 min; OD lecture at 570nm instead of 600 nm.

GLP compliance:

yes

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

Non-animal testing is the default requirement for skin sensitisation.

Details on the study design:

Test item preparation:
The test item was dissolved in DMSO to a final concentration of 200 mM. The compound formed a clear colourless solution at 200 mM. From the stock 12 spike solutions in DMSO were prepared: 2000, 1000, 500, 250, 125, 62,5 31,3 15,6 7.81, 3.91, 1.95 and 0.978 µM.

Test System:
The KeratinoSens cells are transfected HaCaT keratinocytes that include a 56-base-pair insertion containing the ARE sequence from the AKR1C2 gene, a SV40 promoter, the luciferase gene in the vector pGL4 from Promega, and a stable insertion allowing for cells to be selected in the presence of Geneticin (G418).

Cell culture:
- Assay medium: Dulbecco's Modified Eagle Medium (DMEM) containing GlutaMAX I, 1000 mg/L D-Glucose, Sodium Pyruvate (Gibco) supplemented with: 9.1% fetal bovine serum (FBS) (Hyclone).
- Maintenance medium: Dulbecco's Modified Eagle Medium (DMEM) containing GlutaMAX TM I, 1000 mg/L D-Glucose, Sodium Pyruvate (Gibco) supplemented with: 9.1% FBS and 5.5 mL Geniticin (G418) (Invitrogen) (final concentration 500 µg/mL)
- Exposure medium: Dulbecco's Modified Eagle Medium containing GlutaMAX TM I, 1000 mg/L D-Glucose, Sodium Pyruvate supplemented with: 1% FBS

Environmental conditions:
Cells will be exposed to the various dilutions of the test articles for approximately 48 +/- 1 hours in the incubator at standard culture conditions (37 +/- 1°C humidified air containing 5 +/- 1% Co2)

Preparation of the positive control:
The positive control used in the case of KeratinoSensTM is Cinnamic aldehyde. Cinnamic aldehyde will be serially diluted in DMSO to concentrations 100X of the final concentrations for the assay. The 100X DMSO Master Plate stock solutions will be added to the 4X Master Plate containing 1%DMEM to a 4X concentration. Addition of the 4X concentrations to the wells already containing 150 µL 1% DMEM will result in a final 1X concentration. The final concentrations of the positive control will be 6.40, 3.20, 1.60, 0.800 and 0.400 mM.

Subculturing:
The KeratinoSens cells will be subcultured when the flask(s) are 60--90% confluent. The medium will be aspired and the cell sheet will be rinsed twice with 10mL of DPBS containing 0.05% EDTA for approximately 2 minutes. One mL of trypsin/EDTA will be added to each flask to cover the cell sheet. The flask will be incubated at standard culture conditions for 5-10 minutes (or until they became dislodged). When more than 50% of the cells become dislodged, the flask will be rapped sharply against the palm of the hand. When more than 90% of the cells become dislodged, the cells will be each re-suspended in 7mL Maintenance media. An appropriate volume of the trypsinized cells in Maintenance Medium will be added to new flasks for passaging. the KeratinoSens cells will be routinely passaged every 2-5 days

Plating of cells:
For testing, cells were 60-90% confluent. The medium will be aspirated and the cell sheet will be rinsed twice with 10 mL of DPBS containing 0.05% EDTA for approximately 2 mnutes. One mL of trypsin/EDTA will be added to each flask to cover the cell sheet. The flask(s) will be incubated at standard culture conditions for 5-10 minutes (or until they become dislodeged). When more than 50% of the cells become dislodged, the flask will be rapped sharply against the palm of the hand. When more than 90% of the cells become dislodged, the cells will be each re-suspended in 5mL Assay Medium (without G418). The concentration of cells will be determined with a coulter counter or hemacytometer. Cells will be diluted to approximately 10000 cells/mL in Assay Medium.

Treatment of cells:
Cultured cells that will be used in seeding the 96-well plates should be fed fresh Growth Medium the day before subculturing to the plates. Each definitive assay will have 5 plates; 3 white-walled plates will be used for the luciferase endpoint, while 2 clear bottomed plates will be used for the cytotoxicity endpoint. These plates are designated as the 1X plates. Each 96-well plate will be uniquely numbered and labeled with the seeding density, cell type, and date of seeding. Cell suspensions of 10000 cells/mL in the Assay Medium will be prepared. 100 µL of the cell suspension will be added to the designated wells on the 96-well plate (i.e. all wells except well H12, which will be left blank. Using a pipette, 100 µL of Assay Medium will be placed into the well designated as blank (H12). the cells will be incubated for 24 +/- 1 hours at standard culture condition

Luciferase activity measurement:
After 48 hours of exposure each white-walled plate will be allowed to cool to room temperature for 30 minutes, the treatment medium will be decanted from each plate. The cultures will be rinsed with 250µL of CMS-DPBS (room temperature), and the DPBS rinsate will be decanted from the wells.
Fifty microliters of CMF-DPBS will be added to each well. Fifty microliters of Promega ONE-GloTM Reagent will be added to each well and the plates will be allowed to incubate at room temperature in the dark for approximately 5 minutes before being read by the luminometer. The plates will be kept away from sunlight or bright fluorescent light before reading. The plates will be read within 45 minutes of addition of the OneGlo reagent. Each plate will be placed onto the plate tray in the Berthold Detection Systems luminometer, and the luminescence determination will be initiated from an IBM-PC hosting te Windows-based Simplicity TM software. The light intensity in each well will be measured at 565 nm in the form of realtive light units (RLUs).

Cytotoxicity assessment using MTT endpoint:
A 0.59 mg/mL MTT solution will be prepared in 1% DMEM and used within 2 hours. After 48 +/- 1 hours, the clear 96-well plates designated for the MTT endpoint will be removed from the incubator. The test article dilutions will be decanted from the plates.
200 µL of 1% DMEM containing 0.59 mg/mL MTT will be added to each well. The plate will be incubated with a plate seal at standard culture conditions for 4 +/- 0.3 hours. After that the MTT solution will be decanted, the plate will be blotted and 200 µL of 10% SLS will be added to each well. The plate will be covered with a plate seal and incubated at standard culture conditions overnight.
the plate(s) will be placed on a plate shaker and shaken for a least 20 minutes at room temperature. The absorbance at 570 nm (OD570) of each well will be measured with a Molecular Devices Vmax plate reader. The plate reader will be turned on at least 15 minutes prior to use, and will be calibrated according to standard operating procedures. The AUTO MIX setting will be selected. Relative survival will be obtained by comparing the amount of MTT reduction by test article treated groups to the MT reduction by the solvent treated group on the same plate. An IC50 value will be determined for the MTT cytotoxicity plate.

Cytotoxicity assessment using NRU endpoint:
A 33 µg/mL neutral red solution will be prepared and used within 30 minutes. A 1:100 dilution of the stock Neutral Red Solution (3.3 mg/mL) in warm 1% DMEM will be filtered though a 0.45 µm filter. After approximately 48 +/- 1 hours, the clear 96-well plates designated for the NRU will be removed from the incubator. The test article dilutions will be decanted from the plates and the cells will be washed once with 250 µL of DPBS containing Ca++Mg++. 250 µL of the 33µg/mL NR solution will be added to each well. The plate will be incubated at standard culture conditions for approximately 3 +/- 0.1 hours.
After that, the neutral red solution will be decanted.
The plate will be washed once with 250 µL of sterile DPBS containing Ca++Mg++. The rinse will be decanted and the plate will be blotted firmly onto paper towells. 100µL of Neutral Red Solvent will be added to all wells. The plate will be shaken at room temperature for at least 20 minutes.
The plate reader will be turned on at least 15 minutes prior to use and will be calibrated according to standard operating procedures. The AUTO MIX setting will be selected. The absorbance at 550 nm (OD550) of each well will be measured with a Molecular Devices Vmax plate reader. Relative survival will be obtained by comparing the amount of red taken up by test article treated groups to the neutral red taken up by the solvent treated group on the same plate. An IC50 value will be determined for the NRU cytotoxicity plate.

Data analysis:
The following parameters are calculated in the KeratinoSensTM test method:
- The maximal average fold induction of luciferase activity (CImax) value observed at any concentration of the tested chemical and positive control,
- The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained,
- The IC50concentration values for 50% reduction of cellular viability.

Criteria for determination of a valid test
The cinnamic aldehyde as a positive control must be positive, thus the gene induction by the control must cross the 1.5 threshold in at least one dose.
Each assay will be assessed using similir criteria as outlined in the Ring Trial (Natsch et al. 2011): 1) The standard deviation of the viability of the solvent controls for the luciferase plates for all of the definitive assays (9 total plates) will be <20%; 2) The cinnamic aldehyde will be significantly poitive and result in an EC1.5 value less than 64 µM; and 3) The cinnamic aldehyde will produce a 2 to 8 fold induction at 64 µM.

Evaluation of test results
A test article will be considered to have sensitization potential if:1) The EC1.5 value falls below 1000µM (or 200 µg/mL) in at least 2 of 3 repetitions; 2) At the lowest concentration with a gene induction above 1.5, cellular viability should be greater than 70%; and 3) An apparent overall dose response should be similar between repetitions.
Since 2 measures of cytotoxicity will be assessed, varying results in the NRU and MTT endpoit may occur that could affect the 70% viability criteria.

Key result

Run / experiment:

other: mean of the 3 Experiment

Parameter:

other: Imax

Remarks:

EC1.5 = 21.20 µM

Value:

2.53

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

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

ACCEPTANCE OF RESULTS:
- The luciferase activity induction obtained with the positive control, Cinnamic Aldehyde, was above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was < 64 µM after the definitive assays. Cinnamic Aldehyde showed a clear positive effect.
- Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20%.

The test article was tested in three definitive assays. Each definitive assay included a set of 5 plates (3 for gene induction, 2 for cytotoxicity assessment). The test article was tested at 12 concentrations ranging from 2000 to 0.975 µM. The positive control, Cinnamic Aldehyde, was tester at 5 concentrations ranging from 64 to 4 µM.

Summary of the EC1.5 and IC50 of the definitive assays :

IIVS Test Article	Sponsor Designation	EC 1.5 value (µM)	Mean IC50 (µM)		Potential Sensitizer?
			MTT	NRU
Test item	Test item	21.20	186.30	180.93	YES
Cinnamic Aldehyde	Positive Control	11.27	> 64	> 64	YES

Additional luciferase induction information that includes the Imax and the CImax :

IIVS Test Article Number	Sponsor Designation	Maximal Induction (Imax)	Conc. for maximal gene induction (CImax)
Test item	Test item	2.53	125 µM
Cinnamic Aldehyde	positive Control	5.61	64 µM

Interpretation of results:

other: Positive response

Conclusions:

The test item is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) in the KeratinoSensTM assay under the experimental conditions described in the report.

Executive summary:

The test item was evaluated for the ability to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in a KeratinoSens(TM) test similar to the OECD Guideline No. 442D and in compliance with GLP. In this test, KeratinoSens(TM) cells cultures were treated with 12 concentrations of test chemical (0.978, 1.95, 3.91, 7.81, 15.6, 62.5, 125, 250, 500, 1000 and 2000 µM) and control substances (positive control: Cinnamic Aldehyde in DMSO, vehicle control: 1% DMSO in exposure medium, blank) and were then incubated for about 48 hours at 37 +/-1.0°C in presence of 5% CO2. Luciferase activity (Imax and EC1.5) was assessed using a luminometer. The toxicity of the test substance was evaluated with the assessment of cell viability (MTT and NRU test). A total of 3 independent experiments were performed. Care was taken to avoid evaporation of the volatile test compound.

The test item an IC50 of 186.30 µM for MTT and 180.93 µM for NRU . A statistically mean of significant induction of the luciferase activity (EC1.5 value 21.20 μM; p<0.001 Student’s t test) was measured in the three experiment. The maximum luciferase activity induction (Imax) was 2.53-fold at 2000 μM.

Overall, the test item is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed at test concentrations of < 1000 μM with a cell viability of >70% compared to the vehicle control in two out of three experiments. Finally, it is concluded that the KeratinoSensTM assay is valid.

The test item is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) in the KeratinoSensTM assay, thus the test item is predicted to have a potential to induce skin sensitisation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Two in chemico/in vitro key studies were identified :

- In the in chemico assay (Citoxlab, 2014, rel.1), the skin sensitisation potential of the test item was evaluated using a direct peptide binding assay (DPRA) similarly to the OECD Guideline No. 442C and in compliance with GLP.The reactivity of the test item was evaluated by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item at the ratios 1:10 cysteine:test item and 1:50 lysine:test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with ultra-violet detection at 220 nm. Peptide reactivity was reported as percent depletion based on the amount of peptide remaining in the sample relative to the average amount measured in the Quality Control samples.

The acceptance criteria of the samples for the calibration curve, QC and positive control were satisfied. The study was therefore considered to be valid.

The mean of the percentage cysteine and percentage lysine depletions was calculated to be 50.00%. Accordingly, the test item was considered to be highly reactive.

- In the in vitro assay (IIVS, 2014, rel.2), the test item was evaluated for the ability to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in a KeratinoSens(TM) test similar to the OECD Guideline No. 442D and in compliance with GLP. KeratinoSens(TM) cells cultures were treated with test chemical diluted at 0.977, 1.95, 3.91, 7.81, 15.6, 62.5, 125, 250, 500, 1000 and 2000 µM and control substances (positive and vehicle) and were then incubated for about 48 hours at 37 °C. Luciferase activity (Imax and EC1.5) was assessed using a luminometer. The toxicity of the test substance was evaluated with the assessment of cell viability (MTT and NRU test). A total of 3 independent experiments were performed. Care was taken to avoid evaporation of the volatile test compound.

The test item has an IC50 of 186.30 µM for MTT and 180.93 µM for NRU. A statistically mean of significant induction of the luciferase activity (EC1.5 value 21.20 μM; p<0.001 Student’s t test) was measured in the three experiment. The maximum luciferase activity induction (Imax) was 2.53-fold at 2000 μM.

Overall, the test item is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed at test concentrations of < 1000 μM with a cell viability of >70% compared to the vehicle control in two out of three experiments. Finally, it is concluded that the KeratinoSensTM assay is valid. The test item is concluded as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) in the KeratinoSensTM assay and thus it is considered to have a potential to induce skin sensistiation..

The substance was not predicted to be a skin senstiser at 0.01% in a Human repeated patch test (HRL, 2014, n= 110 volunteers) but this does not help for classification purposes (sub-categorisation).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self classification:

Based on the available data, the substance is classified as skin sensitiser Category 1 according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

No direct scientific data are available on the substance to address respiratory sensitisation