Registration Dossier

Administrative data

Description of key information

Skin sensitisation

- In chemico (DPRA)

The test material was considered to have no or minimal peptide reactivity. Therefore, the DPRA prediction is considered as negative and the test material may have no potential to cause skin sensitisation.

- In vitro skin sensitisation (Keratinosens test)

The test material was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

Food and Chemical Toxicology

According to the literature data, the substance (when tested at a concentration of 2 % in petrolatum) produced no sensitisation recations following a maximisation test conducted on 25 volunteers.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 March 2018 to 29 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
yes
Remarks:
please refer to "Principles of method if other than guideline" for further information
Principles of method if other than guideline:
Deviations from the study plan included:
- the test material was directly added to the selected vehicle to reach the concentration of 100 mM in order to avoid the any loss of test material
- all the lysine samples (co-elution controls, reference controls, test material and positive control samples) were incubated up to 29 hours and 38 minutes instead of 24 (± 2) hours. Since the incubation was homogeneous on this lysine analytical sequence and since the acceptance criteria were met, this slight deviation was considered not to have any significant impact on the validity or integrity of the study.

These deviations were considered not to have compromised the validity or integrity of the study.
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
This test is part of a tiered strategy for skin sensitisation assessment, approved by regulatory authorities in the EU.
Test material information:
Composition 1
Specific details on test material used for the study:
As the test material was found to be volatile, it was sampled with a pipette and the respective weight of the sampling was determined using the density of the test material. The test material was directly added to the selected vehicle (acetonitrile) to reach the concentration of 100 mM. This formulation had the aspect of a colorless limpid solution and was used just after its preparation.
Details on study design:
CONTROL ITEMS
- Positive control: Cinnamaldehyde

- Co-elution control samples
In order to detect possible co-elution of the test material with a peptide, co-elution control samples were prepared by incubating the test material formulation with each buffer used to dilute the peptides. Cysteine or lysine peptides were not added to these samples.

- Reference control samples
For each peptide, the analytical batch included reference control samples (sub-categorised in reference control A, B or C samples). All these control samples were prepared in triplicate and at the nominal concentration of 0.500 mM. These samples were used to:
reference control A: check the accuracy of the calibration curve for peptide quantification,
reference control B: check the stability of the peptide during analysis,
reference control C: check that the solvent did not impact the percentage of peptide depletion.

TEST SYSTEMS
- Cysteine peptide
Peptide sequence: AC-RFAACAA-COOH
Peptide sequence synonyms: AC-Arg-Phe-Ala-Ala-Cys-Ala-Ala-COOH
Molecular weight: 750.88 g/mol
Supplier: JPT Peptide Technologies GmbH
Batch No.: 111016HS_MHeW1017
Storage condition: At -20 °C
Description: White powder

The cysteine peptide solution was freshly prepared at 0.667 mM in an aqueous phosphate buffer (pH 7.5) solution.

- Lysine peptide
Peptide sequence: AC-RFAAKAA-COOH
Peptide sequence synonyms: AC-Arg-Phe-Ala-Ala-Lys-Ala-Ala-COOH
Molecular weight: 775.91 g/mol
Supplier: JPT Peptide Technologies GmbH
Batch No.: 120514HSDWW1017
Storage condition: At -20 °C
Description: White powder
Specific handling conditions : None

The lysine peptide solution was freshly prepared at 0.667 mM in an aqueous ammonium acetate buffer (pH 10.2) solution.

METHOD
The test material was tested in one run. The run was processed as described below.

- Preparation of the samples
The following samples were prepared in triplicate except for the co-elution control samples for which only one sample was prepared per peptide buffer.

- Co-elution control samples preparation
For the co-elution control with cysteine peptide:
50 µL of test material 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 material formulation was incubated with 750 µL of lysine peptide dilution buffer (without lysine peptide).

- Reference control samples preparation
Reference control A and B samples:
In a vial, acetonitrile was added to a volume of peptide solution (cysteine or lysine) to achieve a nominal concentration of 0.500 mM.
Reference control C samples:
Reference control C samples were prepared for each solvent used to dissolve the test and positive control items.
For the reference control C prepared with cysteine peptide:
50 µL of vehicle (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 reference control C prepared with lysine peptide:
In parallel, 250 µL of vehicle (acetonitrile) was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate buffer at pH 10.2).

- 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 material samples preparation
For the reactivity of test material with cysteine peptide:
50 µL of test material 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 material with lysine peptide:
In parallel, 250 µL of test material 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 controls, reference controls, test material and positive control samples) were then incubated during 24 (± 2) hours for the cysteine-peptide analytical sequence or up to 29 hours and 35 minutes for the lysine-peptide analytical sequence at 25 °C and protected from light before injection into the HPLC/UV system.
At the end of the incubation period, a visual inspection of the samples was performed prior to HPLC analysis to detect precipitate or phase separation.
Samples presenting precipitate were centrifuged at 400g for a period of 5 minutes at room temperature and only supernatants were then injected onto the HPLC/UV system. Otherwise, the vials were directly 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 each peptide (lysine and cysteine) in separate solutions of 20 % acetonitrile:peptide dilution buffer to obtain at least six different concentration levels ranging from 0.0167 to 0.534 mM. A dilution buffer blank was also included in the standard calibration curve.
The calibration curves were defined by the relationships between the peak area signal of the peptide 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.

HPLC/UV Conditions:
- Analytical column: Zorbax SB C18, 100 x 2.1 mm, 3.5 µm, (Waters). In-line filter C18, 4.0 x 2.0 mm (Phenomenex)
- Mobile phase: Mobile phase A: acetonitrile + 0.085 % TFA; Mobile phase B: milli-Q water + 0.1 % TFA
- Flow: 350 µL/minute
- Gradient:
Time % Mobile phase A % Mobile phase B
0 10 90
10 25 75
11 90 10
13 90 10
13.5 10 90
20 10 90
- UV wavelength: 220 nm
- Rinse solution: acetonitrile
- Oven temperature: 30 °C
- Autosampler temperature: nominal temperature +25 °C
- Injection volumes: 7 µL for the Cysteine-peptide analytical sequence and 3 µL for the Lysine-peptide analytical sequence
- Retention times: Cysteine-peptide: approx. 9.5 minutes; Lysine-peptide: approx. 6.8 minutes
- Total analysis time: 20 minutes

DATA ANALYSIS AND CALCULATION
- Calculation of the percent peptide depletion
Each appropriate peak was integrated and the peak area for calibration standards, control and test material samples were determined. Based on the concentration of standards and their peak area, a linear calibration curve was generated. Then, the concentration of peptide was determined in each sample from absorbance at 220 nm, measuring the peak area of the appropriate peaks and calculating the concentration of peptide using the linear calibration curves. Then, for each positive control and test material replicate, the percent depletion of peptide was determined from the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent) by using the following formula:

% depletion = [1 - (peptide peak area in replicate injection / mean peptide peak area in relevant reference control C samples)] x 100

Then, the mean percent depletion of the three replicates was calculated for each peptide as well as the mean of the percent cysteine and percent lysine depletions. Negative depletion values were considered as "Zero" for the calculation of the mean % depletion.
Peak areas and peptide concentrations are presented in the report. Standard Deviation (SD) and Coefficient of Variation (CV) were calculated and reported.

- Evaluation of the possible co-elution of the test item with the lysine or cysteine peptides
In order to detect possible co elution of the test materials with a peptide, chromatograms of the co-elution control samples were analysed and compared with those of the reference control C samples.

ACCEPTANCE CRITERIA
The run was considered valid if the following criteria were fully met:
- the calibration curve should have a coefficient of determination (r²) ≥ 0.99,
- the mean peptide concentrations of the reference control A samples should be within ± 10 % of the nominal concentration,
- the cinnamaldehyde depletion control samples should meet the following acceptance criteria:
- for the cysteine peptide, the mean percent depletion value should be between 60.8 and 100 % with a SD < 14.9 %,
- for the lysine peptide, the mean percent depletion value should be between 40.2 and 69.0% with a SD < 11.6 %,
- the CV of the mean peptide peak area of the nine reference control B and C samples in acetonitrile must be < 15.0 %,
- the test item’s results were considered valid if the following criteria were fully met:
- the mean peptide concentrations of the reference control C samples prepared in the appropriate solvent should be within ± 10% of the nominal concentration,
- the maximum SD for the test material replicates should be < 14.9 % for the percent cysteine depletion value and < 11.6 % for the percent lysine depletion value.

DATA INTERPRETATION AND CLASSIFICATION
A test material was considered to have the following classification for peptide reactivity according to the mean peptide depletion results for cysteine and lysine:
Mean cysteine and lysine % depletions No co-elution Co-elution with Cys Co-elution with Lys Co-elution with cystein & lysine
0 % ≤ mean %depletion ≤ 6.38 % no/ minimal reactivity Inconclusive apply Cys
6.38 % < mean %depletion ≤ 22.62 % low reactivity ≥ low reactivity 1:10 only inconclusive
22.62 % < mean %depletion ≤ 42.47 % moderate reactivity ≥ moderate reactivity prediction
42.47 % < mean %depletion ≤ 100% high reactivity high reactivity model

Cysteine 1:10-only prediction model:
Mean of cysteine depletion Reactivity class
0 % ≤ Mean % depletion ≤ 13.89 % No or minimal reactivity
13.89 % < Mean % depletion ≤ 23.09 % Low reactivity
23.09 % < Mean % depletion ≤ 98.24 % Moderate reactivity
98.24 % < Mean % depletion ≤ 100 % High reactivity

The result of the DPRA is considered as positive as soon as low reactivity is detected.
Key result
Parameter:
other: percent cysteine and percent lysine depletions
Run / experiment:
mean
Value:
0.58
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
SOLUBILITY RESULTS
The test material was found soluble at 100 mM in acetonitrile without sonication. Acetonitrile was therefore chosen to be vehicle.

EVALUATION OF THE PRESENCE OF PRECIPITATE AT THE END OF THE INCUBATION WITH PEPTIDES
At the end of the incubation period, a visual inspection of all samples (co-elution controls, reference controls, test material and positive control samples) was performed prior to HPLC analysis.
As precipitate was observed in the positive control samples incubated with the cysteine and lysine peptides, these vials were centrifuged at 400g for a period of 5 minutes at room temperature to force precipitate to the bottom of the vial. Thus, only supernatants were injected into the HPLC/UV system.

EVALUATION OF THE RESULTS
The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

Analysis of the chromatograms of the co-elution samples indicated that the test material did not co-elute with either the lysine or the cysteine peptides.
- for the cysteine peptide, the mean depletion value was 0.20 %,
- for the lysine peptide, the mean depletion value was 0.95 %.


Interpretation of results:
other: Not sensitising
Conclusions:
The mean of the percent cysteine and percent lysine depletions was equal to 0.58%. Accordingly, the test material was considered to have no or minimal peptide reactivity. Therefore, the DPRA prediction is considered as negative and the test material may have no potential to cause skin sensitisation.
Executive summary:

The potential for the test material to be sensitising to human skin was investigated in chemico, in a study which was conducted in accordance with the standardised guideline OECD 442C, under GLP conditons.

The reactivity of the test material was evaluated by monitoring peptide depletion following a 24-hour contact between the test material and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with UV detection at 220 nm.

Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent).

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

Analysis of the chromatograms of the co-elution samples indicated that the test material did not co-elute with either the lysine or the cysteine peptides.

-  for the cysteine peptide, the mean depletion value was 0.20 %,

-  for the lysine peptide, the mean depletion value was 0.95 %.

The mean of the percent cysteine and percent lysine depletions was equal to 0.58 %. Accordingly, the test material was considered to have no or minimal peptide reactivity. Therefore, the DPRA prediction is considered as negative and the test material may have no potential to cause skin sensitisation.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 March 2018 to 13 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
This test is part of a tiered strategy for skin sensitisation assessment, approved by regulatory authorities in the EU.
Test material information:
Composition 1
Details on study design:
TEST AND CONTROL ITEMS
- Vehicle and negative control: Based on solubility results, the vehicle was DMSO.
This vehicle was used as the negative control, and was applied to cells at a concentration of 1 % in culture medium.
- Positive control: Cinnamic aldehyde (CA)
For each run, the positive control item was dissolved in DMSO to a final concentration of 200 mM. This solution was then further diluted to a final concentration of 6.4 mM. It was diluted in DMSO by serial dilutions in the Master plate 100x, using a dilution factor of 2, to obtain a total of 5 concentrations. Subsequently, each formulation of the Master plate 100x was diluted 25-fold in treatment medium in another 96-well plate called "Master plate 4x". The final tested concentrations ranged from 4 to 64 µM. All these formulations were prepared within 4 hours before use, then kept at room temperature and protected from light until use.
- Test material formulations: On the basis of solubility results, the test material was diluted in DMSO at 200 mM.
One formulation was prepared for each run. It was then diluted in DMSO by serial dilutions, using a dilution factor of 2 to obtain a total of 12 concentrations in a 96-well plate; this 96 well plate was called "Master plate 100x". Subsequently, each formulation of the Master plate 100x was 25-fold diluted in treatment medium in another 96 well plate called "Master plate 4x" taking care to adjust all wells to the same DMSO level. All formulations were prepared within 4 hours before use, and kept at room temperature and protected from light until use.

TEST SYSTEM
- KeratinoSens cells: the cell line KeratinoSens is stably transfected with a modified plasmid. This plasmid contains an ARE sequence from the AKR1C2 gene and a SV40 promotor which are inserted upstream of a luciferase gene. The resulting plasmid was transfected into HaCaT keratinocytes and clones with a stable insertion selected in the presence of Geneticin / G-418. Induction of luciferase gene is the endpoint evaluated and reflects the activation by the test material of the Nrf2 transcription factor in this test.
- Supplier: this cell line was provided by Givaudan.
- Batch: D1.
- Storage condition: at -80 °C.
- Mycoplasm: absence of mycoplasm was confirmed.

METHOD
The test material was tested in two independent runs using cells from a different passage number. The plates were processed as described below:
- Solubility assay
A solubility assay was performed prior the first treatment in order to select the vehicle (among DMSO, water for injections or treatment culture medium).
Since the test material was found soluble in DMSO at 200 mM, this stock formulation was diluted in treatment culture medium to the final concentration of 2000 µM. Then, a visual inspection of the sample was performed to evaluate the presence or absence of precipitate/emulsion.

- Method for a run of KeratinoSens assay
Cell seeding for testing:
Cells were grown using general culture procedures up to 80-90% confluence. The day prior to treatment, cells were washed twice with D-PBS containing 0.05% EDTA, harvested, re-suspended in maintenance medium and counted using Trypan Blue dye. Cell concentration was adjusted to a density of 8 x 10E4 cells/mL. Cells were then distributed into four 96-well plates (three white plates and one transparent plate), by adding 125 µL (representing 1 x 10E4 cells) per well taking care to avoid sedimentation of the cells during seeding. After seeding, the cells were grown for 24 (± 1) hours in the 96-well microtiter plates prior to test material addition.

- Treatment
After the 24-hour growing period, the medium was removed by aspiration and replaced by 150 µL of treatment medium. From the Master plate 4x, a volume of 50 µL was added to each well of the three white assay plates and 50 µL to the transparent plate for the cytotoxicity evaluation. All plates were covered by a sealing membrane to avoid evaporation of volatile test items and to avoid cross-contamination between wells. The plates were then incubated for 48 (± 2) hours at 37 °C, 5 % CO2, 90 % humidity.

- Endpoint measurements
Microscopic observation to evaluate the presence or absence of precipitate - transparent plate:
After the 48 (± 2) hours incubation period, the presence or absence of precipitate/emulsion was determined in each well by microscopic inspection.

Luminescence flash signal to evaluate induction signal - white plates:
After incubation, the supernatants from the white assay plates were discarded. The cells were washed once with D-PBS. A volume of 20 µL of passive lysis buffer was added to each well and the cells were incubated for 20 (± 2) minutes at room temperature and under orbital shaking. The plates containing the passive lysis buffer were then placed in the luminometer for reading using the following program: 50 µL of the luciferase substrate was added to each well. 1 second after this addition, the luciferase signal was integrated for 2 seconds.

Absorbance signal to evaluate the cytotoxicity - transparent plate:
For the cell viability assay plate, the medium was replaced by 200 µL of treatment medium. A volume of 27 µL of a MTT solution at 5 mg/mL in D-PBS was then added to each well of the transparent 96-well plate. The plates were covered with a sealing membrane and returned at 37 °C in the incubator in humidified atmosphere for 4 hours (± 10 minutes). At the end of the incubation period, the medium was removed and a volume of 200 µL of a 10 % SDS solution was added to each well. The plates were covered with a sealing membrane and placed at 37 °C in the incubator in humidified atmosphere for an overnight period to extract the formazan from cells. After the overnight incubation, the absorption of each well was determined at 600 nm using the plate reader.

RESULTS ANALYSIS
Data evaluation was performed using a validated Excel sheet. The generated raw data (luminescence data for the luciferase activity and absorbance data for the MTT test) were pasted into an Excel template, and all data processing was performed automatically.

For the MTT and the luciferase data, the background value recorded in the empty well without cells (blank) was subtracted.
For the MTT data, the % viability was calculated for each well in the test plate in relation to average of the six negative control wells.
For the luciferase data, the average value of the six negative control wells was set to 1, and for each well in the plate, the fold induction was calculated in relation to this value.

For wells in which a statistically significant gene-induction (using a student test, also called T-test) over the 1.5 threshold was found, the following parameters were calculated from the processed raw data:
- Imax: maximal induction factor of luciferase activity compared to the negative control over the complete dose-response range measured,
- EC1.5: concentration at which a 1.5-fold luciferase gene induction is obtained,
- IC50 and IC30: concentrations effecting a reduction of cellular viability by 50 and 30 %,
- Indication whether significant 1.5-fold gene induction occurred below the IC30.

The data were plotted in graphs and the Imax and the EC1.5 values were visually checked since uneven dose response curves or large variation may lead to wrong extrapolations.

Also, the individual and overall geometric means IC50 and IC30 were calculated, when applicable.

ACCEPTANCE CRITERIA
Each run was considered valid if the following criteria were met:
- the positive control results should be positive, thus the gene induction should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations,
- the average EC1.5 value for the positive control should be within two standard deviations of the historical mean. In addition, the average induction (Imax) in the three replicate plates for the positive control at 64 µM should be between 2 and 8. If the latter criterion was not fulfilled, the dose response of cinnamic aldehyde was carefully checked, and the run was accepted if there was a clear dose-response with increasing luciferase activity at increasing concentrations for the positive control,
- the average coefficient of variation of the luminescence reading in the negative control wells of the triplicate plates should be < 20 %.

EVALUATION CRITERIA
The results of each run were analysed individually and if the test material was classified as positive in two runs, the final outcome was considered positive. If the test material was classified as negative in two runs, the final outcome was negative. In case, the first two runs were not concordant, a third run was performed and the final outcome was that of the two concordant runs.

The test material was considered as positive if the following four conditions are all met in two of two or in two of three runs, otherwise the KeratinoSens prediction is considered as negative:
- the Imax is > 1.5-fold and statistically significantly different as compared to the negative control (as determined by a two-tailed, unpaired Student’s T-test),
- at the lowest concentration with a gene induction > 1.5-fold (i.e. at the EC1.5 determining value), the cell viability is > 70 %,
- the EC1.5 value is < 1000 µM (or < 200 µg/mL for test item without MW),
- there is an apparent overall dose-response for luciferase induction (or a reproducible biphasic response).

Key result
Parameter:
other: Imax
Run / experiment:
Experiment 1
Value:
1.3
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Parameter:
other: Imax
Run / experiment:
Experiment 2
Value:
1.28
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Other effects / acceptance of results:
SOLUBILITY TEST
In the solubility test, the test material was found soluble in DMSO at 200 mM. Therefore, this vehicle was selected for the preparation of the test material stock formulations.
No precipitate or emulsion was observed once the test material stock formulation was diluted in the treatment culture medium to a final concentration of 2000 µM.

KERATINOSENS RUN
- First and second runs
All acceptance criteria were met for the positive and negative controls in each run, both runs were therefore considered as validated. The criterion "the average induction (Imax) in the three replicate plates for the positive control at 64 µM should be between 2 and 8" was not fulfilled (i.e. Imax of 25.63) in the second run. However, since a clear dose response with increasing luciferase activity at increasing concentrations was obtained for the positive control, this was considered not to have any impact on the validity of the results of the second run.

Both runs were performed using the following concentrations 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1 % DMSO.

At these tested concentrations:
- no precipitate/emulsion was observed in any test material-treated wells at the end of the 48-hour treatment period in both runs,
- no noteworthy decrease in cell viability was noted (i.e. cell viability > 70 %), therefore no IC30 or IC50 was calculated in both runs,
- no statistically significant gene-fold induction above the threshold of 1.5 were noted in comparison to the negative control at any tested concentrations. Thus, no EC1.5 was calculated.
Moreover, the Imax value were < 1.5 (i.e. 1.30 and 1.28 in the first and the second run, respectively).

The evaluation criteria for a negative response were met in both runs; the final outcome was therefore negative.
Interpretation of results:
other: Not sensitising
Conclusions:
Under the experimental conditions of this study, the test material was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.
Executive summary:

The potential for the test material to be sensitising to human skin was investigated in vitro, in a study which was conducted in accordance with the standardised guideline OECD 442D, under GLP conditons.

During the study, the KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37 °C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test material and of positive controls. The treated plates were then incubated for 48 hours at 37 °C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Two independent runs were performed.

All acceptance criteria were met for the positive and negative controls in each run, both runs were therefore considered as validated. The criterion "the average induction (Imax) in the three replicate plates for the positive control at 64 µM should be between 2 and 8" was not fulfilled (i.e. Imax of 25.63) in the second run. However, since a clear dose response with increasing luciferase activity at increasing concentrations was obtained for the positive control, this was considered not to have any impact on the validity of the results of the second run.

Both runs were performed using the following concentrations 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1 % DMSO.

At these tested concentrations:

- no precipitate/emulsion was observed in any test material-treated wells at the end of the 48-hour treatment period in both runs,

- no noteworthy decrease in cell viability was noted (i.e. cell viability > 70 %), therefore no IC30 or IC50 was calculated in both runs,

- no statistically significant gene-fold induction above the threshold of 1.5 were noted in comparison to the negative control at any tested concentrations. Thus, no EC1.5 was calculated.

- the Imax value were < 1.5 (i.e. 1.30 and 1.28 in the first and the second run, respectively).

Under the experimental conditions of this study, the test material was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

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

- In chemico skin sensitosation (DPRA)

The potential for the test material to be sensitising to human skin was investigated in chemico, in a study which was conducted in accordance with the standardised guideline OECD 442C, under GLP conditons. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The reactivity of the test material was evaluated by monitoring peptide depletion following a 24-hour contact between the test material and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with UV detection at 220 nm.

Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent).

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

Analysis of the chromatograms of the co-elution samples indicated that the test material did not co-elute with either the lysine or the cysteine peptides.

-  for the cysteine peptide, the mean depletion value was 0.20%,

-  for the lysine peptide, the mean depletion value was 0.95%.

The mean of the percent cysteine and percent lysine depletions was equal to 0.58%. Accordingly, the test material was considered to have no or minimal peptide reactivity. Therefore, the DPRA prediction is considered as negative and the test material may have no potential to cause skin sensitisation.

- In vitro skin sensitisation (Keratinosens test)

The potential for the test material to be sensitising to human skin was investigated in vitro, in a study which was conducted in accordance with the standardised guideline OECD 442D, under GLP conditons. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, the KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37°C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test material and of positive controls. The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Two independent runs were performed.

All acceptance criteria were met for the positive and negative controls in each run, both runs were therefore considered as validated. The criterion "the average induction (Imax) in the three replicate plates for the positive control at 64 µM should be between 2 and 8" was not fulfilled (i.e. Imax of 25.63) in the second run. However, since a clear dose response with increasing luciferase activity at increasing concentrations was obtained for the positive control, this was considered not to have any impact on the validity of the results of the second run.

Both runs were performed using the following concentrations 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO.

At these tested concentrations:

- no precipitate/emulsion was observed in any test material-treated wells at the end of the 48-hour treatment period in both runs,

- no noteworthy decrease in cell viability was noted (i.e. cell viability > 70%), therefore no IC30 or IC50 was calculated in both runs,

- no statistically significant gene-fold induction above the threshold of 1.5 were noted in comparison to the negative control at any tested concentrations. Thus, no EC1.5 was calculated.

- the Imax value were < 1.5 (i.e. 1.30 and 1.28 in the first and the second run, respectively).

Under the experimental conditions of this study, the test material was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

Food and Chemical Toxicology

According to the literature data, the substance (when tested at a concentration of 2 % in petrolatum) produced no sensitisation recations following a maximisation test conducted on 25 volunteers.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to skin sensitisation.