Reaction mass of 1-(3,3-dimethylcyclohexyl)ethanone and 2,6,6-trimethylcycloheptanone

Endpoint summary

Description of key information

Skin sensitization (DPRA: OECD TG 442C): Negative

Skin sensitisation (Keratinosens): OECD TG 442D): Negative

Skin sensitization (h-CLAT: OECD TG 442E): Negative

The substance is negative in all three in vitro assays and therefore the substance is not considered a skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 November, 2016 - 16 December, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

Controls:
Positive control: ethylene dimethacrylate glycol, 31.25-500 µM, tested in triplicate
Negative control: DMSO (vehicle) (1% in exposure medium)
Blank: on each plate three blank wells were tested (no cells and no treatment) to assess background values

Number of replicates: three 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.

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. The compound formed a clear colourless solution at 200 mM. From the stock 11 spike solutions in DMSO were prepared (2-fold dilution series): 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.781, 0.391, 0.195 and 0.0977 mM.
The stock and spike solution were diluted 25-fold with exposure medium resulting in concentrations of 8000, 4000, 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.813 and
3.906 µM (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, 31.25, 15.63, 7.81, 3.91, 1.95 and 0.976 µM. All concentrations of the test item were tested in triplicate. All formulations formed a clear solution. Three independent experiments were performed.

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±1.0oC in the presence of 5% CO2.

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 (e.g. by adding 10% SDS solution to each well) overnight. 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). 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 intepretation:
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 (Figure 1):
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

Acceptance criteria:
• 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).
• 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.
• 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 6 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 3.26 and the EC1.5 31.9 µM.
Experiment 2: the positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.69 and the EC1.5 27.0 µM.
Experiment 3: the positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.75 and the EC1.5 62.8 µM.

Key result

Run / experiment:

other: Experiment 1, 2000 µM

Parameter:

other: Imax

Value:

2.22

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

no dose response was observed

Key result

Run / experiment:

other: Experiment 2, 2000 µM

Parameter:

other: Imax

Value:

1.27

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Experiment 3, 125 µM

Parameter:

other: Imax

Value:

1.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (12.7, 6.2% and 7.3% in experiment 1, 2 and 3 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 (31.9 µM, 27.0 µM and 62.8 µM in experiment 1, 2 and 3 respectively). A dose response was observed in all three experiments and the induction at 250 µM was >2-fold in experiment 1 and 3 and with 1.94-fold just below 2-fold in experiment 2.

- Acceptance criteria met for variability between replicate measurements: Experiment 1 was considered inconclusive. Luciferase induction >1.5-fold was observed in the first experiment at all test concentrations with exception at 1000 µM. The maximum luciferase activity induction (Imax) was 2.22-fold but no clear dose response was observed. No EC1.5 was calculated. The first experiment was concluded as inconclusive. No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in experiment 2 and 3. The maximum induction was 1.27 and 1.10-fold in experiment 2 and 3, respectively. Therefore the test substance is classified as negative in Experiment 2 and 3.

Summary tables

Table 1. Overview luminescence induction and cell viability of the test substance in Experiments 1, 2 and 3

Concentration (µM)	0.98	1.95	3.91	7.81	15.63	31.25	62.50	125	250	500	1000	2000
Exp 1 luminescence	1.63***	1.62***	1.71***	1.69***	1.60***	1.60***	1.67***	1.68***	1.70***	1.74***	1.08	2.22***
Exp 1 viability (%)	101.6	121.4	118.2	119.7	103.1	106.1	108.4	111.7	109.0	113.1	176.5	100.5
Exp 2 luminescence	1.00	0.90	0.94	0.97	0.94	0.97	1.01	1.04	1.01	1.05	1.07	1.27
Exp 2 viability (%)	108.7	115.5	109.8	119.1	136.4	111.1	106.8	102.6	100.8	102.3	92.4	91.3
Exp 3 luminescence	0.92	0.99	1.00	1.03	1.03	1.05	1.03	1.10	1.06	0.94	0.98	1.09
Exp 3 viability (%)	102.0	107.9	93.7	109.3	103.1	66.5	84.8	74.8	78.9	91.5	100.1	87.8

*** p<0.001 Students t-test

Table 2. Overview of luminescence induction and cell viability positive controlEthylene dimethacrylate glycol in Experiments 1, 2 and 3

Concentration (µM)	7.81	15.6	31.3	62.5	125	250
Exp 1 luminescence	1.12	1.20	1.49	1.74***	2.20***	3.26***
Exp 1 viability (%)	86.5	89.2	94.8	96.0	99.7	105.2
Exp 2 luminescence	1.25	1.37	1.55**	1.75***	1.94***	2.69***
Exp 2 viability (%)	122.7	128.1	136.3	148.9	131.7	124.6
Exp 3 luminescence	0.97	1.02	1.23	1.50	1.86***	2.75***
Exp 3 viability (%)	107.3	92.7	92.4	107.1	106.4	107.6

** p<0.01; *** p<0.001 Students t-test

Table 3. Overview of EC1.5, IC30 and IC50 values

	EC1.5 (µM)	Imax	IC30 (µM)	IC50 (µM)
Test substance Experiment 1	NA*	2.22	NA	NA
Test substance  Experiment 2	NA	1.27	NA	NA
Test substance Experiment 3	NA	1.10	NA	NA
Pos Control Experiment 1	31.9	3.26	NA	NA
Pos Control Experiment 2	27.0	2.69	NA	NA
Pos Control Experiment 3	62.8	2.75	NA	NA

N.A. = Not applicable

* No dose response but all test concentrations with exception of 1000 µM showed >1.5-fold induction

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 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. Three independent experiments were performed. The test substance was tested in a concentration range of test concentrations of 0.98 – 2000 µM (2-fold dilution series). Ethylene diemthacrylate glycol was used as a positive control. The acceptance criteria were met. In the first experiment, statistically significant luciferase induction >1.5-fold was observed at all test concentrations with exception at 1000 µM. The maximum luciferase activity induction (I_max) was 2.22-fold but no clear dose response was observed. No EC_1.5 (concentration at which induction of luciferase activity is above 1.5 fold threshold) was calculated. The first experiment was concluded as inconclusive. No biologically relevant induction of the luciferase activity (no EC_1.5value) was measured at any of the test concentrations in experiment 2 and 3. The maximum induction was 1.27 and 1.10-fold in experiment 2 and 3, respectively. Therefore the test substance was considered negative in Experiment 2 and 3. Based on the KeratinoSens^TMprediction model (2 out of 3) it is concluded that the compound is negative in this assay.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 November 2016 - 19 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

d.d. 15/2/2016

Type of study:

direct peptide reactivity assay (DPRA)

Details on the 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 and further diluted in HPLC vials. 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 the 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.

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.

Incubation:
The appearance of the test 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 test 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:

-73.1% depletion (CV 0.71%, n = 3) and -59.8% depletion (CV 1.98%, n = 3) of cysteine and lysine, respectively, was observed with the positive control cinnamic aldehyde.

Key result

Run / experiment:

other: 1

Parameter:

other: cysteine depletion, %

Value:

-0.167

Vehicle controls validity:

valid

Remarks:

stability and precision controls

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 1

Parameter:

other: lysine depletion, %

Value:

-0.108

Vehicle controls validity:

valid

Remarks:

stability and precision controls

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: not applicable. Reference (stability) controls and precision controls of both peptides were met (CV 1.06%, n = 6 and CV 0.47%, n = 6, for cysteine and lysine, respectively, at 0.50 mM).
- Acceptance criteria met for positive control: yes, 73.1% depletion (CV 0.71, n = 3) and 59.8% depletion (CV 1.98%, n = 3) of cysteine and lysine, respectively, was observed with the positive control cinnamic aldehyde.
- Acceptance criteria met for variability between replicate measurements: yes, CV 0.57% and 0.28%, respectively, for cysteine and lysine depletion by the test item.

TEST SUBSTANCE RESULTS:

Mean depletion of -0.167% and -0.108% was observed for the test substance with cysteine and lysine peptides, respectively. No co-elution peaks were observed in either the cysteine or lysine assays. 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 that the test substance was negative in the DPRA and is classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

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. The test substance was within the applicability domain of the assay. Solutions of the test substance were successfully analyzed by the validated DPRA analytical method. The validity criteria were met. There were no co-elution peaks in either cyrsteine or lysine assays. The test substance caused -0.167% cysteine peptide depletion and -0.108% lysine peptide depletion and was classified as “no to minimal reactivity” based on the DPRA prediction model. The substance was thus considered to be negative in the DPRA.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 November, 2016 - 25 November, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD guideline 442E: In vitro skin sensitization: human Cell Line Activation Test (h-CLAT)

Version / remarks:

July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

d.d. 14 September 2015

Type of study:

other: human Cell Line Activation Test (h-CLAT)

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
Solvent: DMSO (final concentration 0.2% in culture medium, also used as a solvent for positive control)
Positive control: DNCB in DMSO diluted with culture medium (2 and 3 μg DNCB/mL)

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 did not exceed 1 × 10^6 cells/mL. The passage numbers of the used THP-1 cells were 9 in both XTT assays and 10 and 11 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) was 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 doses investigated in the main experiment (h-CLAT) were determined with two XTT tests, instead of flow cytometry recommended by the guideline.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. A decrease in number of living cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample. This decrease directly correlates to the amount of orange formazan formed, as monitored by the absorbance. Two independent cytotoxicity experiments were performed with different cell cultures days 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: immediately prior to start the substance was first suspended in culture medium (2.5 mg/mL fine and stable suspension) and further dissolved in culture medium. The maximum concentration of test item was 1250 μg/mL in culture medium, as tested by a solubility test. For the XTT test (dose finding assay) eight concentrations of the test item were analysed. Therefore, dilutions were prepared by 1:2 serial dilutions from the highest suspended/soluble concentration.

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. 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. The second run was cancelled and repeated due to a technical error.
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): 46.6, 55.9, 67.1, 80.5, 96.6, 116.0, 139.2 and 167.0 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 tested 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-aminoactinomycin D (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) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was 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 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 study is considered as valid, if the following criteria are met:
• 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 positive control (DNCB), RFI values of both CD86 and CD54 should exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50%.
• In the DMSO solvent control, RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
• For medium and DMSO controls, the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
• For the test item resulting in negative outcome, the cell viability at the 1.2 × CV75 should be less than 90%. (If the cell viability at the 1.2 × CV75 is more than 90% for a positive tested test item, the data will be acceptable. If 5 mg/mL in saline, 1 mg/mL in DMSO or the highest soluble dose will be used as the maximal test concentration instead of CV75-based dose, the data for test item are accepted independent by the cell viability.)
• The cell viability of at least 4 doses in each experiment should be ≥50%.

Evaluation of results:
The test item is tested in 2 independent runs. If the RFI of CD86 is ≥ 150% or if the RFI of CD54 is ≥ 200% in both independent run data, the test item is considered to be a sensitiser. Otherwise it is considered to be a non-sensitiser. In case of different results in both runs, a third run has to be performed. If the RFI of CD86 is ≥ 150% at any dose in at least 2 of 3 independent run data, or if the RFI of CD54 is ≥ 200% in at least 2 of 3 independent run data, the test item is considered to be a sensitiser. Otherwise it is considered to be a non-sensitiser.

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

Run / experiment:

other: 1, CD86

Parameter:

other: % RFI

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 1, CD54

Parameter:

other: % RFI

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 2, CD86

Parameter:

other: % RFI

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 2, CD54

Parameter:

other: % RFI

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

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%.

Results of the XTT assay:

1st test:

			Microscopic		Photometric
			Evaluation		Evaluation
Test Group	Concentration		Cytotoxicity	Mean Ab-	Standard-	Chem.		Absorbance in % of
	[µg/mL]			sorbance*	Deviation	Blanks		Solvent Control**
Medium Control	-		no	0.773	0.043	0.273		93.19
Solvent Control	-		no	0.818	0.035	0.281		100.00
	9.8		no	0.752	0.081	0.282		87.53
	19.6		no	0.762	0.040	0.287		88.58
	39.1		no	0.734	0.025	0.289		82.81
	78.1		no	0.697	0.026	0.289		76.11
Test Item
	156.3		no	0.617	0.022	0.289		61.10
	312.5		no	0.634	0.037	0.273		67.16
	625		no	0.607	0.064	0.284		60.28
	1250		yes	0.399	0.066	0.302		18.05
Shaded test groups:	cytotoxic effects occurred in the photometric evaluation (< 75% cell viability)

 * mean absorbance (absolute) of 7 wells

** relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium control was 107.4%.

 

The CV75 value of the first XTT test: 83.9 µg/mL

2nd test:

			Microscopic				Photometric
			Evaluation				Evaluation
Test Group	Concentration		Cytotoxicity		Mean Ab-		Standard-		Chem.		Absorbance in % of
	[µg/mL]				sorbance*		Deviation		Blanks		Solvent Control**
Medium Control	-		no	0.697		0.018		0.263			107.67
Solvent Control	-		no	0.678		0.014		0.274			100.00
	9.8		no	0.717		0.028		0.258			113.77
	19.6		no	0.717		0.061		0.236			119.11
	39.1		no	0.691		0.011		0.256			107.70
	78.1		no	0.618		0.018		0.253			90.40
Test Item
			no		0.576		0.018		0.260		78.10
	156.3
	312.5		no		0.524		0.024		0.260		65.40
	625		no		0.456		0.017		0.263		47.86
	1250		yes		0.299		0.019		0.255		10.85
Shaded test groups:	cytotoxic effects occurred in the photometric evaluation (< 75% cell viability)

* mean absorbance (absolute) of 7 wells

** relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium control was 92.9%.

 

The CV75 value of the second XTT test: 194.4 µg/mL

 

The mean CV75 value of both XTT tests: 139.2 µg/mL

Results of the h-CLAT test:

1st test run:

	Concentration (µg/mL)	Antibody	RFI (%)	Cell Viability
				(%)
Medium Control	-	CD 54	100.0	100.0
	-	CD 86	100.0
DMSO Control	-	CD 54	100.0	100.0
	-	CD 86	100.0
Positive Control (DNCB)	2.0	CD 54	494.7*	72.5
		CD 86	327.2*
	3.0	CD 54	507.7*	79.1
		CD 86	393.7*
Test Item	46.6	CD 54	100.8	94.9
		CD 86	116.0
	55.9	CD 54	97.6	95.7
		CD 86	111.2
	67.1	CD 54	88.2	95.5
		CD 86	101.6
	80.5	CD 54	94.5	93.5
		CD 86	128.8
	96.6	CD 54	105.5	92.4
		CD 86	126.4
	116.0	CD 54	96.1	99.6
		CD 86	120.8
	139.2	CD 54	114.2	91.9
		CD 86	133.6
	167.0	CD 54	116.5	76.3
		CD 86	129.6

*  RFI value of CD86 or CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

2nd test run:

	Concentration (µg/mL)	Antibody	RFI (%)	Cell Viability (%)
Medium Control	-	CD 54	100.0	100.0
	-	CD 86	100.0
DMSO Control	-	CD 54	100.0	100.0
	-	CD 86	100.0
Positive Control (DNCB)	2.0	CD 54	225.9*	66.7
		CD 86	536.8*
	3.0	CD 54	326.7*	64.9
		CD 86	451.5*
Test Item	46.6	CD 54	94.2	90.9
		CD 86	76.7
	55.9	CD 54	102.3	89.8
		CD 86	95.0
	67.1	CD 54	101.2	90.5
		CD 86	95.6
	80.5	CD 54	100.0	87.5
		CD 86	69.8
	96.6	CD 54	102.3	86.6
		CD 86	70.4
	116.0	CD 54	126.7	86.1
		CD 86	61.6
	139.2	CD 54	146.5	85.0
		CD 86	71.7
	167.0	CD 54	126.7	78.5
		CD 86	54.1

*  RFI value of CD86 or CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

Interpretation of results:

other: h-CLAT was negative

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 hour. The test substance falls within the applicability domain of the assay. The validity criteria 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 HTT tests:46.6, 55.9, 67.1, 80.5, 96.6, 116.0, 139.2 and 167.0 µg/mL.The test substance was tested in 2 valid independent runs.The RFI of CD86 and CD54 wasbelow 150% and 200%, respectively, at all tested dose levels of both independent run data. Therefore, the test item is considered to be a non-sensitizer based on the information of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Below the in vitro skin sensitisation assays are presented

DPRA

In the DPRA assay, conducted according to OECD TG 442C, the test substance caused -0.167% and -0.108% depletion of cysteine and lysine peptides, respectively. Based on this, the substance was classified as "no to minimal reactivity" and considered to be a non-sensitizer.

In the h-CLAT test, conducted according to OECD TG 442E, the test substance was tested in two independent runs at concentrations of 46.6, 55.9, 67.1, 80.5, 96.6, 116, 139.2 and 167μg/mL. The acceptance criteria were met. The RFI values of CD86 and CD54 were below 150% and 200%, respectively, at all tested concentrations in both experiments. Therefore the test item was considered to give a negative response in the h-CLAT assay.

Keratinosens

In the KeratinoSens Assay (ARE-Nrf2 luciferase reporter assay), conducted according to OECD TG 442D, three independent experiments were conducted. In the first experiment, statistically significant luciferase induction >1.5-fold was observed at all test concentrations with exception at 1000 µM. The maximum luciferase activity induction (I_max) was 2.22-fold but no clear dose response was observed. No EC_1.5 (concentration at which induction of luciferase activity is above 1.5 fold threshold) was calculated. The first experiment was concluded as inconclusive. No biologically relevant induction of the luciferase activity (no EC_1.5value) was measured at any of the test concentrations in experiment 2 and 3. The maximum induction was 1.27 and 1.10-fold in experiment 2 and 3, respectively. Therefore the test substance was considered negative in Experiment 2 and 3. Based on the KeratinoSens^TMprediction model (2 out of 3) it is concluded that the compound is negative in this assay.

h-CLAT

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 hour. The test substance falls within the applicability domain of the assay. The validity criteria 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 HTT tests: 46.6, 55.9, 67.1, 80.5, 96.6, 116.0, 139.2 and 167.0 µg/mL.The test substance was tested in 2 valid independent runs.The RFI of CD86 and CD54 was below 150% and 200%, respectively, at all tested dose levels of both independent run data. Therefore, the test item is considered to be a non-sensitizer based on the information of this test.

Justification for classification or non-classification

Based on the information above: Negative in DPRA, Keratinosens and h-CLAT in vitro skin sensitisation assays the substance does not need to be classified for skin sensitisation according to EU CLP (EC No. 1272/2008 and its updates).