Registration Dossier

Administrative data

Description of key information

Skin sensitisation (OECD TG 442C, DPRA): negative, no indication for skin sensitisation

Skin sensitization (OECD TG 442D, KeratinoSens): negative, no indication for skin sensitisation

Skin sensitization (OECD TG 442E, h-CLAT): positive, indication for skin sensitisation

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:
10 March 2017 - 17 March 2017
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))
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
Information from in vitro/in chemico test method(s) are recognised according to Article 13(3) of regulation (EC) No 1907/2006.
Test material information:
Composition 1
Details on study design:
Skin sensitisation (In chemico test system) - Details on study design:
Synthetic peptide containing cysteine: Ac-RFAACAA-COOH, lot number 1556171, purity 95% (by HPLC), supplied by AnaSpec, stored frozen (-10°C to -30°C).
Synthetic peptide containing lysine: Ac-RFAAKAA-COOH, lot number 1556172, purity 94% (by HPLC), supplied by AnaSpec, stored frozen (-10°C to -30°C).
Positive control: cinnamic aldehyde, purity > 95%, was prepared at a concentration of 100 mM in acetonitrile.

Preparation of peptide stock solutions:
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (for Cysteine, 100 mM phosphate buffer pH 7.5, for Lysine 100 mM Ammonium acetate buffer pH 10.2).

Preparation of peptide calibration standards:
Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was also prepared.

Preparation of Reference (Stability) Controls and Precision Controls:
Reference (stability) controls and precision controls of both peptides were prepared at a concentration of 0.5 mM in acetonitrile. These were injected throughout the analytical run to confirm consistency of peptide response throughout each analytical run.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls:
A 100 mM solution in acetonitrile of the test substance was prepared.
Cysteine Peptide Depletion Samples (in triplicate) were prepared by dilution of the 100 mM test substance solution in more acetonitrile and Cysteine peptide stock solution. The final sample concentration was 5 mM of test substance, 0.5 mM Cysteine.
In place of the test substance, the positive control solution contained cinnamic aldehyde at a concentration of 5 mM with 0.5 mM cysteine.
The co-elution control sample contained 5 mM of the test substance in phosphate buffer solution. An additional control sample of 5 mM of test substance in acetonitrile was also prepared so as to positively identify peak(s) of the test item in the co-elution control ensuring that the test item had not evaporated away during incubation and injection of the samples on the HPLC.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls:
A 100 mM solution in acetonitrile of the test substance was prepared and further diluted in HPLC vials.
Lysine Peptide Depletion Samples (in triplicate) were prepared by dilution of the 100 mM test substance solution in Lysine peptide stock solution. The final sample concentration was 25 mM of the test substance, 0.5 mM Lysine.
In place of the test substance, the positive control solution contained cinnamic aldehyde at a concentration of 25 mM with 0.5 mM lysine.
The co-elution control sample contained 25 mM of the test substance in ammonium acetate buffer solution. An additional control sample of 25 mM of test substance in acetonitrile was also prepared so as to positively identify peak(s) of the test item in the co-elution control ensuring that the test item had not evaporated away during incubation and injection of the samples on the HPLC.

Incubation:
The appearance of the substance, positive control samples and co-elution controls in the HPLC vials was documented following preparation with the vials then placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.

Analysis:
The concentration of both the cysteine and lysine peptides in the presence of the substance and the associated positive controls were quantified by HPLC using UV detection.
Equipment: HPLC Waters Alliance 2695 separation module and 2487 dual wavelength detector.
Column: Agilent Zorbax SB C18, 3.5 μm, 100 × 2.1 mm
Guard column: Phenomenex AJO4286
Column temperature: 30 °C
Sample temperature: 25 °C
Mobile phase A: 0.1% trifluoroacetic acid in water
Mobile phase B: 0.085% trifluoroacetic acid in acetonitrile
Flow rate: 0.35 mL/minute
Detector wavelength: UV, 220 nm
Injection volume: 2 μL
Run time: 30 minutes
Approximate retention time (cysteine): 11 minutes
Approximate retention time (lysine): 7 minutes
Calculations:
The peak area response for each peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration. The area responses of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured. Peptide depletion was determined using the following equation:

% peptide depletion = 100 - [(Peptide peak area in replicate depletion samples x 100) / (Mean peptide peak area of reference (stability) control samples)]

Acceptance criteria for analytical measurements are presented in Table 1 in the section "Any other information on materials and methods incl. tables". Information on the interpretation of the results is presented in the same section in Table 2.
Positive control results:
72.5% depletion (SD 0.11%, n = 3) and 59.7% depletion (SD 1.40%, n = 3) of cysteine and lysine, respectively, was observed with the positive control cinnamic aldehyde.
Key result
Parameter:
other: cysteine depletion, %
Run / experiment:
1
Value:
0.633
Vehicle controls valid:
yes
Remarks:
stability and precision controls
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: lysine depletion, %
Run / experiment:
1
Value:
0.046
Vehicle controls valid:
yes
Remarks:
stability and precision controls
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: not applicable. Acceptance criteria for reference (stability) controls and precision controls of both peptides were met (CV 0.70%, n = 6 and CV 1.17%, n = 6, for cysteine and lysine, respectively, at 0.51 mM).
- Acceptance criteria met for positive control: yes, 72.5% depletion (SD 0.11%, n = 3) and 59.7% depletion (SD 1.40%, n = 3) of cysteine and lysine, respectively, was observed with the positive control cinnamic aldehyde.
- Acceptance criteria met for variability between replicate measurements: yes, SD 0.40% and 0.91%, respectively, for cysteine and lysine depletion by the test item.

TEST SUBSTANCE RESULTS:
Mean depletion of 0.633% and 0.0460% was observed for the test substance with cysteine and lysine peptides, respectively. A small co-elution peak, equivalent to 0.086% of the mean peak area of the Lysine peptide was observed in the Lysine assay. This small co-elution peak in the Lysine assay did not have an impact on the data. No co-elution peak was observed in the Cysteine assay.
Based on these results the reactivity is classified as "no to minimal".
Interpretation of results:
other: DPRA was negative
Conclusions:
It can be concluded that this DPRA test is valid, and with the mean result of 0.340% depletion the test substance is placed in the reactivity class of “no to minimal reactivity” and thus is predicted to be negative in the DPRA.
Executive summary:

In a GLP-compliant OECD guideline 442C study, Direct Peptide Reactivity Assay (DPRA) was used to assess the reactivity and sensitizing potential of the test substance. Solutions of the test substance (5 mM substance with 0.5 mM Cysteine and 25 mM substance with 0.5 mM Lysine) were successfully analyzed by the validated DPRA analytical method. All analytical acceptance criteria of the test were met. A small co-elution peak, equivalent to 0.086% of the mean peak area of the Lysine peptide was observed in the Lysine assay. This small co-elution peak in the Lysine assay did not have an impact on the data. No co-elution peak was observed in the Cysteine assay. The substance caused 0.633% cysteine peptide depletion and 0.0460% lysine peptide depletion. Based on the mean result of 0.340% depletion the substance is classified as “no to minimal reactivity” based on the DPRA prediction model. The substance was thus considered to be negative in the DPRA.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 May 2017 - 02 June 2017
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)
Version / remarks:
February, 2015
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
Information from in vitro/in chemico test method(s) are recognised according to Article 13(3) of regulation (EC) No 1907/2006.
Test material information:
Composition 1
Details on study design:
Skin sensitisation (In vitro test system) - Details on study design:

Controls:
Positive control: ethylene dimethacrylate glycol, 7.81-250 μM (final concentration DMSO of 1%), tested in triplicate
Negative control: DMSO (vehicle) (1% in exposure medium), eighteen wells were tested per plate.
Blank: on each plate three blank wells were tested (no cells and no treatment) to assess background values

Number of replicates: two independent experiments, each concentration tested in triplicate for the luciferase activity measurements, one parallel replicate for MTT cell viability assay.

Test system:
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.
Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages.
One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator.

Experimental conditions:
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 63 – 96 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.7 – 37.4°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Test item preparation:
No correction was made for the composition/purity of the test item.
The test item was dissolved in DMSO to a final concentration of 200 mM (clear colourless solution). The 100-fold dilution in DMEM of 200 mM showed no precipitation (final concentration 2000 μM). This concentration was selected as highest concentration for the main assay.
In the first experiment, the test item was dissolved in DMSO to a final concentration of 200 mM (clear colourless solution). From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium (final concentration DMSO of 4%). These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.91, 1.95 and 0.98 μM (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution. No precipitation was observed at the start and end of the treatment in the 96-well plates.
In the second experiment, the test item was dissolved in DMSO to a final concentration of 200 mM (clear colourless solution). From this stock 11 spike solutions in DMSO were prepared (1.25-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium (final concentration DMSO of 4%). These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1600, 1280, 1024, 819, 655, 524, 419, 336, 268, 215 and 172 μM (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution. No precipitation was observed at the start and end of the treatment in the 96-well plates.
Test item concentrations were used within 2.5 hours after preparation. Any residual volumes were discarded.

Media:
Basic medium
Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.
Maintenance medium
Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum and geneticin (500 μg/ml).
Exposure medium
Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.

Treatment of cells:
The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control substances were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours at 37±1.0°C in the presence of 5% CO2. In total 2 experiments were performed.

Luciferase acitivity measurement:
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 μL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the luminometer to assess the quantity of luciferase (integration time one second).

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; CAS No. 298-93-1) and cells were incubated for 3 hours at 37°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 is measured with the TECAN Infinite® M200 Pro Plate Reader.

Data analysis:
The following parameters are calculated in the KeratinoSensTM test method:
• The maximal average fold induction of luciferase activity (Imax) 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 IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.
Fold luciferase activity induction is calculated by Equation 1, and the overall maximal fold induction (Imax) is calculated as the average of the individual repetitions.
Equation 1: Fold induction= (Lsample - Lblank)/(Lsolvent - Lblank)
Where:
Lsample is the luminescence reading in the test chemical well
Lblank is the luminescence reading in the blank well containing no cells and no treatment
Lsolvent is the average luminescence reading in the wells containing cells and solvent (negative) control

The EC1.5 is calculated by linear interpolation according to Equation 2, and the overall EC1.5 is calculated as the mean of the individual repetitions.

Equation 2: EC1.5 = (Cb - Ca) x [(1.5 - Ia) / (Ib - Ia)] + Ca
Where:
Ca is the lowest concentration in μM with > 1.5 fold induction
Cb is the highest concentration in μM with < 1.5 fold induction
Ia is the fold induction measured at the lowest concentration with > 1.5 fold induction (mean of three replicate wells)
Ib is the fold induction at the highest concentration with < 1.5 fold induction (mean of three replicate wells)

Viability is calculated by Equation 3:
Equation 3: Viability = 100 x [(Vsample - Vblank) / (Vsolvent - V blank)]
Where:
Vsample is the MTT-absorbance reading in the test chemical well
Vblank is the MTT-absorbance reading in the blank well containing no cells and no treatment
Vsolvent is the average MTT-absorbance reading in the wells containing cells and solvent (negative) control
Control IC50 and IC30 are calculated by linear interpolation, and the overall IC50 and IC30 are calculated as the mean of the individual repetitions.

In case the luciferase activity induction is larger than 1.5 fold, statistical significance is shown by using a two-tailed Student’s t-test, comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05).
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data. The lowest concentration with > 1.5 fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.

Data interpretation
A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 μM (or < 200 μg/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction

Negative results obtained with concentrations <1000 μM or 200 μg/mL should be considered as inconclusive.

Acceptance criteria:
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.81 to 250 μM).
b) The EC1.5 should be between 5 and 125 μM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.
Positive control results:
Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 5.01 (at 250 μM) and the EC1.5 22.8 μM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.99 (at 250 μM) and the EC1.5 27.0 μM.
Key result
Parameter:
other: Imax
Run / experiment:
Experiment 1
Value:
1.62
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: Experiment 1, Imax at 2000 μM
Remarks:
first, second and fourth criterion of positive result are met
Key result
Parameter:
other: EC1.5
Remarks:
μM
Run / experiment:
Experiment 1
Value:
1 400
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: Experiment 1,
Remarks:
third criterion of positive result is not met, therefore Experiment 1 is negative
Key result
Parameter:
other: Imax
Run / experiment:
Experiment 2
Value:
1.25
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: Experiment 2, Imax at 2000 μM, no EC1.5 could be calculated
Remarks:
first criterion of positive result is not met, therefore Experiment 2 is negative
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative (solvent) control: yes. The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (9.6% and 7.1% in experiment 1 and 2, respectively).
- Acceptance criteria met for positive control: yes. The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration. The EC1.5 of the positive control was between 5 and 125 μM (22.8 μM and 27.0 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (5.01-fold and 3.99-fold in experiment 1 and 2, respectively).
- Acceptance criteria met for variability between replicate measurements: The test substance showed no toxicity, in both experiments the viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated. The maximum luciferase activity induction (Imax) was 1.62-fold and 1.25-fold in experiment 1 and 2 respectively. In the first experiment, a dose related induction of the luciferase activity was measured. The calculated EC1.5 value was 1400 μM. Since the EC1.5 is ≥ 1000 μM the test item is classified as negative in the first experiment. No induction of the luciferase activity (no EC1.5 value) was measured in the second experiment. Since this negative result was observed at test concentrations ≥ 1000 μM with a cell viability of >70% the test item is also classified as negative in the second experiment.

Summary tables

Table1 Overview Luminescence Induction and Cell Viability of the substance in Experiment 1 and 2

Concentration (µM)

0.977

1.95

3.91

7.81

15.6

31.3

62.5

125

250

500

1000

2000

Exp 1 luminescence

0.69

0.73

0.80

0.85

0.89

0.93

0.96

1.03

1.19

1.35

1.42

1.62***

Exp 1 viability (%)

80.9

81.5

87.9

94.3

92.5

76.4

97.5

90.9

94.8

84.1

90.4

75.3

Concentration (µM)

172

215

268

336

419

524

655

819

1024

1280

1600

2000

Exp 2 luminescence

0.75

0.88

0.91

0.94

1.03

1.10

1.09

1.18

1.15

1.23

1.24

1.25

Exp 2 viability (%)

100.2

101.5

104.2

98.2

85.6

96.6

91.5

92.5

97.2

97.3

93.7

105.0

***p<0.001 Student’s t-test

Table2 Overview Luminescence Induction and Cell Viability Positive Control EDMG in Experiment 1 and 2

Concentration (µM)

7.81

15.6

31.3

62.5

125

250

Exp 1 luminescence

1.14

1.33

1.70***

2.43***

3.03***

5.01***

Exp 1 viability (%)

101.5

105.2

93.4

90.7

94.5

101.7

Exp 2 luminescence

1.09

1.31

1.57***

2.00***

3.02***

3.99***

Exp 2 viability (%)

101.0

103.1

107.6

120.9

125.2

144.4

***p<0.001 Student’s t-test

Table3 Overview EC1.5, Imax, IC30and IC50Values

 

EC1.5 (µM)

Imax

IC30(µM)

IC50(µM)

Test item Experiment 1

1400

1.62

NA

NA

Test item Experiment 2

NA

1.25

NA

NA

Pos Control Experiment 1

22.8

5.01

NA

NA

Pos Control Experiment 2

27.0

3.99

NA

NA

NA = Not applicable

Interpretation of results:
other: KeratinoSens assay was negative.
Conclusions:
In a GLP-compliant guideline study, the test substance did not cause a biologically relevant induction in luciferase activity in the Keratinosens assay. Based on this, the test substance is considered to give a negative result under the experimental conditions in this assay.
Executive summary:

The GLP-compliant in vitro KeratinoSens (ARE-Nrf2 luciferase reporter) assay was performed in accordance with OECD guideline 442D to assess the skin sensitising potential of the test substance. Two independent experiments were performed. In the first experiment the test substance was tested in a concentration range of 0.977 – 2000 μM (2-fold dilution series). Due to induction of the luciferase activity the test concentrations for the second experiment were 172-2000 μM (1.25-fold dilution series). Ethylene dimethacrylate glycol was used as a positive control. The acceptance criteria of the test were met. The substance showed no toxicity (no IC30 and IC50 value) at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.62-fold and 1.25-fold in experiment 1 and 2 respectively. In the first experiment, an induction of the luciferase activity was measured. The calculated EC1.5 value was 1400 μM. Since the EC1.5 is ≥ 1000 μM the test item is classified as negative in the first experiment. No induction of the luciferase activity (no EC1.5 value) was measured in the second experiment. Since this negative result was observed at test concentrations ≥ 1000 μM with a cell viability of >70% the test item is also classified as negative in the second experiment. Based on the KeratinoSensTM prediction model it is concluded that the substance is negative in the KeratinoSensTM assay.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 February 2017 - 10 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: OECD 442E; In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)
Version / remarks:
July 2016
Deviations:
yes
Remarks:
The cytotoxicity measurement and estimation of the CV75 value of the dose finding assay is performed by XTT test instead of flow cytometry.
GLP compliance:
yes (incl. certificate)
Type of study:
other: human Cell Line Activation Test (h-CLAT)
Justification for non-LLNA method:
Information from in vitro/in chemico test method(s) are recognised according to Article 13(3) of regulation (EC) No 1907/2006.
Test material information:
Composition 1
Details on study design:
Skin sensitisation (In vitro test system) - Details on study design:
Medium control and Solvent control for the Test Item: Culture medium
Positive control: DNCB in DMSO diluted with culture medium (2 and 3 μg DNCB/mL).
Solvent control for the positive control (h-CLAT): DMSO (final concentration 0.2%).
Cell line: THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC, #TIB-202. THP-1 cells are used as surrogate for human myeloid dendritic cells and show enhanced CD86 and/or CD54 expression when treated with sensitisers. The cell density should not exceed 1 × 10^6 cells/mL. The passage numbers of the used THP-1 cells was 11 in both XTT assays and 12 and 17 in the h-CLAT for runs 1 and 2, respectively.
Culture medium: RPMI-1640 supplemented with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate, 1% (v/v) L-glutamine and appropriate antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin) is used to culture the cells during the assay.
Preparation and seeding of THP-1 cells: On the day of the cytotoxicity experiment (XTT) directly before the application of the test item, solvent and medium control, a volume of 100 μL with a cell density of 0.9 - 1 × 10^6 THP-1 cells/mL was seeded in each well of a 96-well flat bottom plate. For the main experiment (h-CLAT) 0.9 - 1 × 10^6 cells/well in a volume of 500 μL was seeded in a 24-well plate before the treatment.

Dose finding assay: The test item concentrations investigated in the main experiment (h-CLAT) were determined with two XTT tests. The XTT test is based on the cleavage of the yellow tetrazolium salt XTT [= (sodium 3'-(1-phenylaminocarbonyl) - (3,4 - tetrazolium) – bis - (4 – methoxy – 6 - nitro) - benzenesulfonic acid hydrate)] to form an orange water soluble formazan dye by dehydrogenase activity in active mitochondria. This method was first described 1988 by SCUDIERO et al. and improved in subsequent years by several other investigators.
Two independent cytotoxicity experiments were performed with different cell cultures to obtain a reliable CV75. The mean of two CV75 values was used to determine the dose-range for the main experiment (h-CLAT).
CV75 is defined as the concentration of toxicant required to reduce the relative absorbance to 75% of the solvent control and is calculated as:
CV75 = Conc.>75 - [((Conc.>75 - Conc.<75) x (%>75 - 75))/(%>75 - %<75)], where:
a) Conc.>75 = maximal measured concentration with the % of solvent control > 75%
b) Conc.<75 = minimal measured concentration with the % of solvent control < 75%
c) %>75 = relative absorpbance at a) in %
d) %<75 = relative absorpbance at b) in %
Test item preparation: On the day of the experiment (immediately prior to start) the test item was solved in culture medium. The maximum concentration of test item was 5000 μg/mL (culture medium, fine and stable suspension), as tested by a solubility test. For the XTT test (dose finding assay) eight concentrations of the test item were analysed. For this, dilutions were prepared by 1:2 serial dilutions from 5000 μg/mL culture medium.

XTT Labelling Mixture: The XTT labelling mixture consists of two components, a XTT buffer solution and the substrate solution. Both components were mixed right before application at a ratio of 1:100.

Treatment: After the cell seeding, 100 μL of the test item dilutions, the medium and solvent controls, respectively were added to the cells. All dose groups were tested in 7 replicates for each XTT test. At the end of the incubation period of 24 ± 1 hours, the cell cultures were microscopically evaluated for morphological alterations.

XTT Labelling and Measurement: At the end of the incubation period, 50 μL of the XTT labelling mixture were added to each well. The cells were incubated and subsequently transferred to a microplate reader (Versamax® Molecular Devices). The absorbance was measured at 450 nm (reference wavelength 690 nm).

Acceptability criteria of XTT assay:
The XTT test is considered to be acceptable if it meets the following criteria:
• mean absorbance of the medium control is ≥ 0.5
• mean viability of the solvent control is ≥ 90% in comparison to the medium control

Main test:
The test item was tested in two independent runs.
Test item preparation: For the test item exposure the highest dose solution calculated from the XTT assay was prepared corresponding to 1.2 × CV75. Further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment. The following concentrations of the test item (solved in culture medium) were tested in the main experiment (h-CLAT): 164.2, 197.1, 236.5, 283.8, 340.6, 408.7, 490.4 and 588.5 μg/mL.

Treatment of the cells: Each volume (500 μL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 1 hours. Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).

Staining of the cells: The triplicates of each test item-treated and not test item treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250 × g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended and blocked with 600 μL of blocking solution at 2 - 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 μL FACS buffer. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control).
All solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis procedure. The cells were gently mixed by hand and incubated light protected for 30 ± 5 min. at 2 - 8 °C (on ice).

Sample preparation for measurement: After staining with the antibodies, the cells were washed twice (2 - 8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 μL of a 7-AAD solution were added.
Flow cytometry acquisition: The expression of cell surface antigens (CD54, CD86) was analyzed by flow cytometry. The FITC acquisition channel (FL-1) should be set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) were set for the optimal detection of DNA-bound 7-AAD fluorescence signal.
Acquisition: A total of 10,000 living cells were analyzed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. The other tubes were acquired without changing the settings of the cytometer. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) was not calculated, if the cell viability was less than 50 % (due to diffuse labelling of cytoplasmic structures that are generated due to cell membrane destruction).
Data analysis and interpretation:
The RFI is used as an indicator of CD86 and CD54 expression, and is calculated as follows for each concentration of every chemical:
RFI (%) = 100 x [(MFI of test item treated cells - MFI of test item treated isotope control cells) / (MFI of solvent control cells - MFI of solvent isotope control cells)], where MFI is geometric mean fluorescent
intensity
The cell viability is calculated as follows:
Cell viability (%) = 100 x [(Mean cytotoxicity of solvent control cells) / (Mean cytotoxicity of the test item treated cells)], where Mean cytotoxicity is the mean of geometric mean (7-AAD) isotype control, geometric mean (7-AAD) CD54 and geometric mean (7-AAD) CD86.

Acceptability criteria of the h-CLAT assay:
The following acceptance criteria should be met when using the h-CLAT method:
• Cell viability of medium control is adjusted to 100% and the cell viability of the DMSO control should be more than 90% in comparison to the medium control.
• In the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
• For both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
• In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50%.
• For the test chemical, the cell viability should be more than 50% in at least four tested concentrations in each run.
Negative results are acceptable only for test items exhibiting a cell viability of < 90% at the highest concentration tested (i.e. 1.2 × CV75). If the cell viability at 1.2 × CV75 is ≥ 90% the negative result should be discarded. In such a case it is recommended to try to refine the dose selection by repeating the CV75 determination. It should be noted that when 5000 μg/mL in saline (or medium or other solvents/vehicles), 1000 μg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test chemical, a negative result is acceptable even if the cell viability > 90%.

Evaluation of results:
For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs:
− The RFI of CD86 is ≥ 150% at any tested concentration (with cell viability ≥ 50%);
− The RFI of CD54 is ≥ 200% at any tested concentration (with cell viability ≥ 50%).
Otherwise, the h-CLAT prediction is considered NEGATIVE.
Based on the above, if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered POSITIVE and a third run does not need to be conducted. Similarly, if the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE without the need for a third run. If however, the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 and the other is only positive for CD54, a third run is required. If this third run is negative for both markers, the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker or for both markers, the h-CLAT prediction is considered POSITIVE.
Positive control results:
The RFI values of the positive controls (DNCB) for CD86 and CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability was >50%.
Key result
Parameter:
other: % RFI, CD86 Antibody
Run / experiment:
1
Value:
> 150
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
positive indication of skin sensitisation
Remarks:
First run
Key result
Parameter:
other: % RFI, CD54 Antibody
Run / experiment:
1
Value:
> 200
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
positive indication of skin sensitisation
Remarks:
First run
Key result
Parameter:
other: % RFI, CD86 Antibody
Run / experiment:
2
Value:
> 150
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
positive indication of skin sensitisation
Remarks:
Second run
Key result
Parameter:
other: % RFI, CD54 Antibody
Run / experiment:
2
Value:
> 200
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
positive indication of skin sensitisation
Remarks:
Second run
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: In the DMSO solvent control, RFI values compared to the medium control of both CD86 and CD54 did not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
- Acceptance criteria met for positive control: The RFI values of the positive controls (DNCB) for CD86 and CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability was >50%.

- Cell viability of the DMSO control was more than 90% in comparison to the medium control.
- For both medium and DMSO control, the MFI ratio of CD86 and CD54 to isotype control was > 105%.
- For the test chemical, the cell viability was more than 50% in at least four tested concentrations in each run.

Results of the XTT assay:

1st test:

 

Microscopic Evaluation

Photometric Evaluation


Test Group

Concen-tration
[µg/mL]

Cytotoxicity

Mean Ab-sorbance*

Standard-Deviation

Chem. Blank

Mean Ab-sorbance – Chemical Blank

Absorbance in % of Solvent Control**

Medium Control

-

no

0.661

0.026

0.218

0.444

95.11

Solvent Control

-

no

0.683

0.039

0.216

0.467

100.00

Test Item

39.1

no

0.693

0.038

0.216

0.477

102.18

78.1

no

0.692

0.042

0.215

0.476

102.09

156.3***

no

1.056

0.065

0.275

0.780

167.21

312.5

no

0.612

0.032

0.215

0.397

85.06

625

no

0.514

0.049

0.216

0.298

63.94

1250

yes

0.226

0.008

0.214

0.013

2.68

2500P

yes

0.227

0.006

0.219

0.008

1.80

5000P

yes

0.227

0.006

0.219

0.008

1.72

Shaded test groups: cytotoxic effects occurred in the photometric evaluation (< 75% cell viability)

P             precipitation

*            mean absorbance (absolute) of 7 wells

**          relative absorbance [rounded values]

***        results were excluded from the evaluation due to a technical error.

The mean viability of the solvent control in comparison to the medium control was 105.14%.

The CV75 value of the first XTT test: 461.4 µg/mL

2nd test:

 

Microscopic Evaluation

Photometric Evaluation


Test Group

Concen-tration
[µg/mL]

Cytotoxicity

Mean Ab-sorbance*

Standard-Deviation

Chem. Blank

Mean Ab-sorbance – Chemical Blank

Absorbance in % of Solvent Control**

Medium Control

-

no

0.550

0.019

0.208

0.342

92.14

Solvent Control

-

no

0.578

0.028

0.206

0.372

100.00

Test Item

39.1

no

0.570

0.037

0.205

0.365

98.35

78.1

no

0.584

0.036

0.205

0.378

101.81

156.3

no

0.559

0.043

0.210

0.349

94.04

312.5

no

0.532

0.039

0.208

0.324

87.16

625

no

0.466

0.065

0.210

0.256

68.78

1250

yes

0.223

0.007

0.208

0.015

4.04

2500P

yes

0.220

0.004

0.211

0.010

2.58

5000P

yes

0.233

0.010

0.213

0.020

5.45

Shaded test groups: cytotoxic effects occurred in the photometric evaluation (< 75% cell viability)

P             precipitation

*            mean absorbance (absolute) of 7 wells

**          relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium control was 108.53%.

The CV75 value of the second XTT test: 519.2 µg/mL

The mean CV75 value of both XTT tests: 490.3 µg/mL

Results of the h-CLAT Test:

First run:

 

Concentration (µg/mL)

RFI (%)
CD 54 Antibody

RFI (%)
CD 86 Antibody

Cell Viability (%)

 

Medium Control

-

100.0

100.0

100.0

DMSO Control

-

100.0

100.0

100.0

Positive Control (DNCB)

2.0

241.3*

590.4*

55.5

3.0

334.6*

591.6*

59.4

Test Item

164.2

239.0*

96.7

82.1

197.1

325.6*

157.2*

81.2

236.5

328.0*

181.6*

72.9

283.8

357.3*

212.5*

64.9

340.6

281.7*

146.1

61.9

408.7

346.3*

238.2*

51.0

490.4

507.3*

389.5*

39.6

588.5

502.4*

480.9*

29.0

Shaded test groups:           cell viability below 50%, are excluded from the evaluation

*            RFI value of CD86 or CD54 fulfilled the positive criteria (CD86150% and CD54200%).

Second run:

 

Concentration (µg/mL)

RFI (%)
CD 54 Antibody

RFI (%)
CD 86 Antibody

Cell Viability (%)

 

Medium Control

-

100.0

100.0

100.0

DMSO Control

-

100.0

100.0

100.0

Positive Control (DNCB)

2.0

213.2*

392.0*

78.0

3.0

200.4*

316.7*

61.0

Test Item

164.2

138.0

119.0

95.2

197.1

132.9

130.6

91.5

236.5

200.4*

134.5

89.1

283.8

142.7

126.7

86.9

340.6

168.2

145.3

89.0

408.7

171.0

153.5*

84.2

490.4

178.0

165.9*

75.9

588.5

167.5

195.0*

66.9

*   RFI value of CD86 or CD54 exceeded the positive criteria (CD86150% and CD54200%).

Interpretation of results:
other: h-CLAT was positive
Conclusions:
In a GLP-compliant guideline study, the test substance was found to be positive in the in vitro h-CLAT test, indicating a possible skin sensitizing potential of the substance.
Executive summary:

The GLP-compliant in vitro Human Cell Line Activation Test (h-CLAT) was performed in accordance with OECD guideline 442E to assess the skin sensitising potential of the test substance dissolved in culture medium when administered to THP-1 cells for 24 ± 1 hours. The validity criteria of the test were met, e.g. the values obtained for controls. The following concentrations of the test item (solved in culture medium) were tested in the main experiment (h-CLAT) based on the results of two XTT tests: 164.2, 197.1, 236.5, 283.8, 340.6, 408.7, 490.4 and 588.5 μg/mL. The test substance was tested in 2 valid independent runs. The RFI of CD86 and CD54 was above 150% and 200%, respectively, in at least one dose in both independent run data. Therefore, the test substance is considered to have an indication of a skin sensitising potential.

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

The skin sensitization potential is assessed with in silico, in vitro and additional information and is shortly summarized. Thereafter the executive summaries of the individual in vitro studies are presented.

Summary of information:

In silico: The substance contains an ethynyl group which is considered to be an electrophilic group. However, this group is not considered to have a protein binding potential in the substance, as the substance does not have a protein binding potential according to the OECD Toolbox and no alert for skin sensitisation is presented by Derek Nexus. In addition, for the hydrolysis product of the substance, 1-ethynylcyclohexanol (Cas no. 78 -27 -3) also containing an ethynyl group, a LLNA is available which shows negative results (ECHA-dissemination site).

In vitro: The substance tested was negative in the DPRA, negative in the KeratinoSens but positive in the h-CLAT. In absence of protein binding (DPRA) and specific signals for skin sensitisation (KeratinoSens), the potential of forming antibodies (h-CLAT) in isolation, is not likely to present a skin sensitization potential.

Additional information: Two HRIPT studies are available for the substance. When tested at a dose of 2% none of the 39 subjects showed a reaction (IFF, 1971). In a more recent study no effects were noted in 107 subjects when a dose of 0.2% was applied (IFF, 2014).

Overall conclusion: The test substance does not have a skin sensitization potential based on the key information of three in vitro skin sensitization tests. The ITS proposed by Bauch et al. 2012, presents that when 2/3 in vitro results are negative the substance can be considered a non-skin sensitiser. In addition, the OECD Toolbox and Derek Nexus predicted no alerts for skin sensitisation. Reference: Bauch, C., Kolle, S.N., Ramirez, T., Eltze, T., Fabian E., Mehling, A., Teubner, W., van Ravenzwaay, B., Landsiedel, R., 2012, Putting the parts together: Combining in vitro methods to test for skin sensitizing potentials, Regul. Toxicol. Pharmacol., 63, 489–504.

DPRA (Direct Peptide Reactivity Assay, OECD TG 442C):

In a GLP-compliant OECD guideline 442C study, Direct Peptide Reactivity Assay (DPRA) was used to assess the reactivity and sensitizing potential of the test substance. Solutions of the test substance (5 mM substance with 0.5 mM Cysteine and 25 mM substance with 0.5 mM Lysine) were successfully analyzed by the validated DPRA analytical method. All analytical acceptance criteria of the test were met. A small co-elution peak, equivalent to 0.086% of the mean peak area of the Lysine peptide was observed in the Lysine assay. This small co-elution peak in the Lysine assay did not have an impact on the data. No co-elution peak was observed in the Cysteine assay. The substance caused 0.633% cysteine peptide depletion and 0.0460% lysine peptide depletion. Based on the mean result of 0.340% depletion the substance is classified as “no to minimal reactivity” based on the DPRA prediction model. The substance was thus considered to be negative in the DPRA.

KeratinoSens (OECD TG 442D):

The GLP-compliant in vitro KeratinoSens (ARE-Nrf2 luciferase reporter) assay was performed in accordance with OECD guideline 442D to assess the skin sensitising potential of the test substance. Two independent experiments were performed. In the first experiment the test substance was tested in a concentration range of 0.977 – 2000 μM (2-fold dilution series). Due to induction of the luciferase activity the test concentrations for the second experiment were 172-2000 μM (1.25-fold dilution series). Ethylene dimethacrylate glycol was used as a positive control. The acceptance criteria of the test were met. The substance showed no toxicity (no IC30 and IC50 value) at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.62-fold and 1.25-fold in experiment 1 and 2 respectively. In the first experiment, an induction of the luciferase activity was measured. The calculated EC1.5 value was 1400 μM. Since the EC1.5 is ≥ 1000 μM the test item is classified as negative in the first experiment. No induction of the luciferase activity (no EC1.5 value) was measured in the second experiment. Since this negative result was observed at test concentrations ≥ 1000 μM with a cell viability of >70% the test item is also classified as negative in the second experiment. Based on the KeratinoSensTM prediction model it is concluded that the substance is negative in the KeratinoSensTM assay.

h-CLAT (human Cell Line Activation Test, OECD TG 442E):

The GLP-compliant in vitro Human Cell Line Activation Test (h-CLAT) was performed in accordance with OECD guideline 442E to assess the skin sensitising potential of the test substance dissolved in culture medium when administered to THP-1 cells for 24 ± 1 hours. The validity criteria of the test were met, e.g. the values obtained for controls. The following concentrations of the test item (solved in culture medium) were tested in the main experiment (h-CLAT) based on the results of two XTT tests: 164.2, 197.1, 236.5, 283.8, 340.6, 408.7, 490.4 and 588.5 μg/mL. The test substance was tested in 2 valid independent runs. The RFI of CD86 and CD54 was above 150% and 200%, respectively, in at least one dose in both independent run data. Therefore, the test substance is considered to have an indication of a skin sensitising potential.

Justification for classification or non-classification

Based on the presented information the substance is not a skin sensitiser. Therefore classification of this substance for skin sensitisation is not warranted according to EU CLP Regulation (EC) No. 1272/2008 and its updates.