Thiamine hydrochloride

Endpoint summary

Description of key information

DPRA, Reinen (2017): Under the conditions of this study the test material was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Keratinosens, Westerink (2017): Under the conditions of this study, the test material is classified as negative as it showed no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes.

U-SENS, Eurlings (2018): Under the conditions of this study, the test material is classified as negative (no increase in the expression levels of CD86 cell surface marker in the U937 cell line).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 July 2017 to 07 July 2017

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))

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Recommended test system in the international OECD guidelines.

Specific details on test material used for the study:

- No correction for the purity/composition of the test material was performed.
- Solubility of the test material in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test material completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvents were evaluated: acetonitrile (ACN) and Milli-Q water (MQ).
- Test material stock solutions were prepared freshly for each reactivity assay.
- For both the cysteine and lysine reactivity assay 55.14 mg of the test material was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1635 μL MQ (tap water purified by reversed osmosis and subsequently passed over activated carbon and ion exchange cartridges) to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test material was dissolved. The test material, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.
- Any residual volumes were discarded.

Details on the study design:

TEST SYSTEM
- Test System: Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL.
- Source: JPT Peptide Technologies GmbH, Berlin, Germany.
- Storage: The peptides were stored in the freezer (≤-15 °C) for a maximum of 6 months.

PREPARATION OF SOLUTIONS FOR CYSTEINE REACTIVITY ASSAY
- Synthetic Peptide Containing Cysteine (SPCC) Stock Solution: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
- SPCC Reference Control Solutions: Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCC stock solution with 250 μL ACN. In addition, a RCcysCwater sample was included to evaluate the effect of the solvent that was used to dissolve the test material on the Percent Peptide Depletion. The RCcysCwater sample was prepared by mixing 750 μL of the 0.667 mM SPCC stock solution with 200 μL ACN and 50 μL MQ.

- SPCC Calibration Curve: A SPCC calibration curve was prepared:
STDcys1 solution (0.534 mM): 1600 μL stock solution of 0.667 mM SPCC + 400 μL CAN
STDcys2 solution (0.267 mM): 1 mL STDcys1 + 1 mL STDcys7
STDcys3 solution (0.133 mM): 1 mL STDcys2 + 1 mL STDcys7
STDcys4 solution (0.067 mM): 1 mL STDcys3 + 1 mL STDcys7
STDcys5 solution (0.033 mM): 1 mL STDcys4 + 1 mL STDcys7
STDcys6 solution (0.017 mM): 1 mL STDcys5 + 1 mL STDcys7
STDcys7 solution (0 mM): 8 mL phosphate buffer (pH 7.5) + 2 mL CAN

- Co-elution Control, Test Material and Positive Control Samples: The co-elution control (CC) samples, test material samples and the cinnamic aldehyde positive control samples (PC) were prepared:
Co-elution control (CC) (1 replicate: 1 CCcys-208708/A): 750 μL Phosphate buffer pH 7.5, 200 μL ACN and 50 μL 208708/A test solution (100 mM)
Cinnamic aldehyde (PC) (3 replicates: PCcys-1 to PCcys-3): 750 μL Stock solution of 0.667 mM SPCC, 200 μL ACN and 50 μL Cinnamic aldehyde solution (100 mM in ACN)
Test material 208708/A (3 replicates: 208708/A-cys-1 to 208708/A-cys-3): 750 μL Stock solution of 0.667 mM SPCC, 200 μL ACN and 50 μL 208708/A test solution (100 mM).

PREPARATION OF SOLUTIONS FOR LYSINE REACTIVITY ASSAY
- Synthetic Peptide Containing Lysine (SPCL) Stock Solution: A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10 mg of SPCL in 19.31 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.
- SPCL Reference Control Solutions: Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL ACN. In addition, a RClysCwater sample was included to evaluate the effect of the solvent that was used to dissolve the test material on the Percent Peptide Depletion. The RClysCwater sample was prepared by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL MQ.

- SPCL Calibration Curve: A SPCL peptide calibration curve was prepared:
STDlys1 solution (0.534 mM): 1600 μL stock solution of 0.667 mM SPCL + 400 μL ACN
STDlys2 solution (0.267 mM): 1 mL STDlys1 + 1 mL STDlys7
STDlys3 solution (0.133 mM): 1 mL STDlys2 + 1 mL STDlys7
STDlys4 solution (0.067 mM): 1 mL STDlys3 + 1 mL STDlys7
STDlys5 solution (0.033 mM): 1 mL STDlys4 + 1 mL STDlys7
STDlys6 solution (0.017 mM): 1 mL STDlys5 + 1 mL STDlys7
STDlys7 solution (0 mM): 8 mL ammonium acetate buffer (pH 10.2) + 2 mL CAN

- Co-elution Control, Test Material and Positive Control Samples: The co-elution control (CC) samples, test material samples and the cinnamic aldehyde positive control samples (PC) were prepared:
Co-elution control (CC) (1 replicate: CClys-208708/A): 750 μL Ammonium acetate buffer pH 10.2 and 250 μL 208708/A test solution (100 mM)
Cinnamic aldehyde (PC) (3 replicates: PClys-1 to PClys-3): 750 μL Stock solution of 0.667 mM SPCL and 250 μL Cinnamic aldehyde solution (100 mM in ACN)
Test material 208708/A: (3 replicates: 208708/A-lys-1 to 208708/A-lys-3): 750 μL Stock solution of 0.667 mM SPCL and 250 μL 208708/A test solution (100 mM)

SAMPLE INCUBATIONS
- After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test material samples) were placed in the autosampler in the dark and incubated at 25±2.5 °C. The incubation times between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample were 24 hours and 23.5 hours, respectively. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
- Prior to HPLC-PDA analysis the samples were visually inspected for precipitation.

HPLC-PDA ANALYSIS
SPCC and SPCL peak areas in the samples were measured by HPLC-PDA. Sample analysis was performed using the following system:

System 1 (used for Cysteine Reactivity Assay):
- Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
- MPS 3C autosampler (DaVinci, Rotterdam, The Netherlands)
- LC Column oven 300 (Thermo Scientific)
- Surveyor PDA detector (Thermo Scientific)

System 2 (used for Lysine Reactivity Assay):
- Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
- HTC PAL autosampler (DaVinci, Rotterdam, The Netherlands)
- Column Oven #151006 (Grace, Worms, Germany)
- Surveyor PDA detector (Thermo Scientific)

- Mobile phase: A: 0.1 % (v/v) TFA in Milli-Q water, B: 0.085 % (v/v) TFA in CAN
- Gradient: 0 min 10 % B, 10 min 25 % B, 11 min 90 % B, 13 min 90 % B, 13.5 min 10 % B and 20 min 10 % B.
- Flow: 0.35 mL/min
- Injection volume: 4 μL
- Sample tray temperature: Set at 25 °C
- Column: Zorbax SB-C18, 100 mm x 2.1 mm, df = 3.5 μm (Agilent Technologies, Santa Clara, CA, USA)
- Guard column: SecurityGuard™ cartridge for C18, 4 x 2.0 mm (Phenomenex, Torrance, CA, USA)
- Column temperature: Set at 30 °C
- Detection: Photodiode array detection, monitoring at 220 and 258 nm

ACCEPTABILITY CRITERIA
The following criteria had to be met for a run to be considered valid:
- The standard calibration curve had to have an r^2>0.99.
- The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8 and 100 % for SPCC and between 40.2 and 69.0 % for SPCL.
- The maximum standard deviation (SD) for the positive control replicates had to be <14.9 % for the Percent Cysteine Peptide Depletion and <11.6 % for the Percent Lysine Peptide Depletion.
- The mean peptide concentration of Reference Controls A had to be 0.50±0.05 mM.
- The Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN had to be <15.0 %.

The following criteria had to be met for a test material’s results to be considered valid:
- The maximum SD for the test material replicates had to be <14.9 % for the Percent Cysteine Depletion and <11.6 % for the Percent Lysine Depletion.
- The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50±0.05 mM.

ANALYSIS
- Data Evaluation: The concentration of SPCC or SPCL was photometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards. The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:
Percent Peptide Depletion = [ 1 – (Peptide Peak Area in Replicate Injection (at 220 nm) / Mean Peptide Peak Area in Reference Controls (at 220 nm))] x 100
In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample, a ratio in the range of 90 %
- Data Interpretation: The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test material. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model, the threshold of 6.38 % average peptide depletion was used to support the discrimination between a skin sensitiser and a non-sensitiser.

Negative DPRA prediction:
0 % ≤ Mean % depletion ≤ 6.38 % (No or minimal reactivity)

Positive DPRA prediction:
6.38 % < Mean % depletion ≤ 22.62 % (Low reactivity)
22.62 % < Mean % depletion ≤ 42.47 % (Moderate reactivity)
42.47 % < Mean % depletion ≤ 100 % (High reactivity)

Positive control results:

Cysteine Reactivity Assay: The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 72.9 ± 2.2 %. This was within the acceptance range of 60.8 to 100 % with a SD that was below the maximum (SD <14.9 %).

Lysine Reactivity Assay: The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 58.5 ± 0.9 %. This was within the acceptance range of 40.2 to 69.0 % with a SD that was below the maximum (SD <11.6 %).

Key result

Run / experiment:

other: Cysteine Reactivity Assay

Parameter:

other: The mean Percent SPCC Depletion

Value:

7

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: ± 5.4 %

Key result

Run / experiment:

other: Lysine Reactivity Assay

Parameter:

other: The mean Percent SPCL Depletion

Value:

5.9

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: ± 2.7 %

Other effects / acceptance of results:

SOLUBILITY ASSESSMENT OF THE TEST MATERIAL
- At a concentration of 100 mM, the test material was not soluble in ACN, but was soluble in MQ. Therefore this solvent was used to dissolve the test material in this DPRA study.


CYSTEINE REACTIVITY ASSAY

Acceptability:
- The correlation coefficient (r^2) of the SPCC standard calibration curve was 0.991. Since the r^2 was >0.99, the SPCC standard calibration curve was accepted.
- The mean peptide concentration of Reference Controls A was 0.480±0.005 mM, the mean peptide concentration of Reference Controls C was 0.490±0.008 mM and the mean peptide concentration of Reference Controls Cwater was 0.479±0.023 mM. The means of Reference Control samples A, and C and Cwater were all within the acceptance criteria of 0.50±0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (MQ) used to dissolve the test material did not impact the Percent SPCC Depletion.
- The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 3.1 %. This was within the acceptance criteria (CV <15.0 %) and confirms the stability of the HPLC run over time.
- The mean area ratio (A220/A258) of the Reference Control samples was 18.69. The mean A220/A258 ratio ± 10 % range was 16.82-20.55. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
- The Percent SPCC Depletion of the positive control cinnamic aldehyde was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 72.9 ± 2.2 %. This was within the acceptance range of 60.8 to 100 % with a SD that was below the maximum (SD <14.9 %).

Results for the Test Material:
- No precipitate was observed in any of the test material samples.
- In the CC sample no peak was observed at the retention time of SPCC, this demonstrated that there was no co-elution of the test material with SPCC. For the 208708/A-cys samples, the mean SPCC A220/A258 area ratio was 18.98. Since this was within the 16.82-20.55 range, this again indicated that there was no coelution of the test material with SPCC.
-The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls Cwater. The mean Percent SPCC Depletion for the test material was 7.0 ± 5.4 %.


LYSINE REACTIVITY ASSAY

Acceptability:
- The correlation coefficient (r^2) of the SPCL standard calibration curve was 0.997. Since the r^2 was >0.99, the SPCL standard calibration curve was accepted.
- The mean peptide concentration of Reference Controls A was 0.506±0.008 mM, the mean peptide concentration of Reference Controls C was 0.502±0.012 mM and the mean peptide concentration of Reference Controls Cwater was 0.494±0.005 mM. The means of Reference Control samples A, and C and Cwater were all within the acceptance criteria of 0.50±0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (MQ) used to dissolve the test material did not impact the Percent SPCL Depletion.
- The CV of the peptide areas for the nine Reference Controls B and C was 2.0 %. This was within the acceptance criteria (CV <15.0 %) and confirms the stability of the HPLC run over time.
- The mean area ratio (A220/A258) of the Reference Control samples was 14.95. The mean A220/A258 ratio ± 10% range was 13.46-16.45. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
- The Percent SPCL Depletion of the positive control cinnamic aldehyde was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 58.5 ± 0.9 %. This was within the acceptance range of 40.2 to 69.0 % with a SD that was below the maximum (SD <11.6 %).

Results for the Test Material:
- No precipitate was observed in any of the samples.
- In the CC sample no peak was observed at the retention time of SPCL, this demonstrated that there was no co-elution of the test material with SPCL. For the 208708/A-lys samples, the mean SPCL A220/A258 area ratio was 15.13. Since this was within the 13.46-16.45 range, this again indicated that there was no coelution of the test material with SPCL.
- The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls Cwater. The mean Percent SPCL Depletion for Thiamine hydrochloride was 5.9 ± 2.7%.

DPRA PREDICTION AND REACTIVITY CLASSIFICATION
- In the cysteine reactivity assay the test material showed 7.0 % SPCC depletion while in the lysine reactivity assay the test material showed 5.9 % SPCL depletion. The mean of the SPCC and SPCL depletion was 6.5 % and as a result the test material was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. Therefore, the test material was considered to be positive in the DPRA.

Table 1: SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for The Test Material

Treatment	SPCC Depletion		SPCL Depletion		Mean of SPCC and SPCL depletion	DPRA prediction and reactivity classification
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
Test Material	7.0 %	± 5.4 %	5.9 %	± 2.7 %	6.5 %	Positive: Low reactivity

Table 2: Acceptability of the Direct Peptide Reactivity Assay (DPRA)

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPCL
Correlation coefficient (r^2) standard calibration curve	>0.99	0.991	>0.99	0.997
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.480 ± 0.005	0.50 ± 0.05	0.506 ± 0.008
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.490 ± 0.008	0.50 ± 0.05	0.502 ± 0.012
Mean peptide concentration RC-Cwater samples (mM)	0.50 ± 0.05	0.479 ± 0.023	0.50 ± 0.05	0.494 ± 0.005
CV (%) for RC samples B and C	<15.0	3.1	<15.0	2.0
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	72.9	40.2-69.0	58.5
SD of peptide depletion cinnamic aldehyde (%)	<14.9	2.2	<11.6	0.9
SD of peptide depletion for the test material	<14.9	5.4	<11.6	2.7

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation.

Interpretation of results:

other: Positive in the DPRA (Low reactivity class)

Conclusions:

Under the conditions of this study the test material was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 442C, under GLP conditions.

The objective of this study was to determine the reactivity of the test material towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test material with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test material to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

Milli-Q water (MQ) was found to be an appropriate solvent to dissolve the test material and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study.

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A, C and Cwater, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test material, were all within the acceptability criteria for the DPRA.

In the cysteine reactivity assay the test material showed 7.0 % SPCC depletion while in the lysine reactivity assay the test material showed 5.9 % SPCL depletion. The mean of the SPCC and SPCL depletion was 6.5 % and as a result the test material was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Under the conditions of this study the test material was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Reference 2

Endpoint:

skin sensitisation: in vitro

Remarks:

KeratinoSens assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 September 2017 to 29 September 2017

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:

2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin sensitisation tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD).

Specific details on test material used for the study:

- No correction was made for the composition/purity of the test material.
- A solubility test was performed. The test material was suspended in DMSO to a final concentration of 200 mM. The test material was vortexed and sonicated (25 min with a maximum temperature of 25.0 °C. The 100-fold dilution of the 200 mM DMSO stock in DMEM formed a clear solution (2000 μM). This concentration was selected as highest concentration for the main assay (highest dose required in the current guideline).
- In the main experiments the test material was suspended in dimethyl sulfoxide (DMSO) at 200 mM. The stock was vortexed and sonicated (10 min with a maximum temperature of 29.0 °C in experiment 1 and for 15 min with a maximum temperature of 28 °C in experiment 2). From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). At concentrations of 50 mM and higher the test material formed a suspension in DMSO whereas at 25 mM and lower it was fully soluble. The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM (final concentration DMSO of 1 %). All concentrations of the test material were tested in triplicate.
- No precipitation was observed at the start and end of the incubation period in the 96-well plates.
- Test material concentrations were used within 2.5 hours after preparation.
- Any residual volumes were discarded.

Details on the study design:

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). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switserland). 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 from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

CELL CULTURE
- 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. 
- Environmental conditions: All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100 % (actual range 29 – 99 %), containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 35.4 – 46.9 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.
- Sub-culturing: Cells were sub-cultured 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 from the frozen stock.

EXPERIMENTAL DESIGN
- Plating of Cells: For testing, cells were 80-90 % confluent. 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. The passage number used was 20 in experiment 1 and 22 in experiment 2.
- 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 material and controls 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 valid experiments were
performed. Initially, experiment 1 did not pass all the acceptability criteria and therefore this part of the study was repeated.
- Luciferase Activity Measurement: The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilised) 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 two seconds).
- Cytotoxicity Assessment: For the KeratinoSens 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 was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The KeratinoSens test is considered acceptable if it meets the following 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 7.8 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 18 wells tested. If the variability is higher, results should be discarded.

INTERPRETATION
- Data analysis: The following parameters are calculated in the KeratinoSens 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 reepetitions.
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 = (Vsample - Vblank) / (Vsolvent - Vblank) × 100
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.
Equation 4: ICx = (Cb – Ca) x [ ((100−x)- Va) / (Vb−Va) ] + Ca
x is the % reduction at the concentration to be calculated (50 and 30 for IC50 and IC30)
Ca is the lowest concentration in μM (or µg/mL) with > x% reduction in viability
Cb is the highest concentration in μM (or µg/mL) with < x% reduction in viability
Va is the % viability at the lowest concentration with > x% reduction in viability
Vb is the % viability at the highest concentration with < x% reduction in viability

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 KeratinoSens 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 KeratinoSens prediction is considered negative:
- 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)
- 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)
- The EC1.5 value is less than (<) 1000 μM (or < 200 µg/mL for test chemicals with no defined MW)
- 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

Positive control results:

- 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 (107 μM and 29 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.56-fold and 3.48-fold in experiment 1 and 2, respectively).

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: Imax

Value:

1.05

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: EC1.5 could not be calculated

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: Imax

Value:

1.07

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: EC1.5 could not be calculated

Other effects / acceptance of results:

EXPERIMENT 1
- No precipitation was observed at the start and end of the incubation period in the 96-well plates.
- The test material showed no toxicity. The viability of the cells was higher than 70 % at all test concentrations and therefore no IC30 and IC50 values could be calculated.
- No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test material. The Imax was 1.05 and therefore no EC1.5 could be calculated.
- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.56 and the EC1.5 107 μM.

EXPERIMENT 2
- No precipitation was observed at the start and end of the incubation period in the 96-well plates.
- The test material showed no toxicity. The viability of the cells was higher than 70 % at all test concentrations and therefore no IC30 and IC50 values could be calculated.
- No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with Thiamine hydrochloride. The Imax was 1.07 and therefore no EC1.5 could be calculated.
- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.48 and the EC1.5 29 μM.

ACCEPTANCE CRITERIA
Both tests passed the acceptance criteria:
- 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 (107 μM and 29 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.56-fold and 3.48-fold in experiment 1 and 2, respectively).
- Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20 % (8.2 and 9.9 % in experiment 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

DISCUSSION
- The test material showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments.
- The maximum luciferase activity induction (Imax) was 1.05-fold and 1.07-fold in experiment 1 and 2 respectively.
- The test material is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations ≥ 1000 μM.

Table 1: Overview of Luminescence Induction and Cell Viability of Test Material in Experiment 1 and 2

Concentration (µM)	0.98	2.0	3.9	7.8	16	31	63	125	250	500	1000	2000
Exp 1 luminescence	0.96	1.05	0.99	0.96	1.01	0.99	1.03	1.05	0.99	1.00	0.94	0.95
Exp 1 viability (%)	99.5	95.1	107.4	93.7	95.5	88.8	130.8	87.5	90.4	99.5	101.2	105.4
Exp 2 luminescence	0.94	0.79	0.95	0.90	0.97	0.93	0.90	0.95	0.94	1.01	1.07	0.96
Exp 2 viability (%)	101.4	109.3	109.1	100.3	98.0	96.0	97.1	93.8	98.0	97.8	117.0	117.7

Table 2: Overview of Luminescence Induction and Cell Viability of Positive Control EDMG in Experiment 1 and 2

Concentration (µM)	7.8	16	31	63	125	250
Exp 1 luminescence	1.00	1.04	1.20	1.33	1.57^***	2.56^***
Exp 1 viability (%)	93.0	112.1	146.1	104.3	109.2	117.2
Exp 2 luminescence	1.34	1.32	1.54***	1.83***	2.93^***	3.48^***
Exp 2 viability (%)	104.4	111.1	115.4	115.9	117.7	115.9

^***p<0.001 Student’s t test

Interpretation of results:

other: Not classified in accordance with EU Criteria

Conclusions:

Under the conditions of this study, the test material is classified as negative as it showed no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes.

Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD TG 442D, under GLP conditions.

The objective of this study was to evaluate the ability of the test material to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay.

The test material was suspended in dimethyl sulfoxide at 200 mM. From this stock 11 spike solutions in dimethyl sulfoxide were prepared. At concentrations of 50 mM and higher the test material formed a suspension in dimethyl sulfoxide whereas at 25 mM and lower it was fully soluble. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 μM (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. No precipitate was observed at any dose level tested. Two independent experiments were performed.

Both experiments passed the acceptance criteria: 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 (107 μM and 29 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.56-fold and 3.48-fold in experiment 1 and 2, respectively); Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20 % (8.2 and 9.9 % in experiment 1 and 2, respectively). Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

The test material showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.05-fold and 1.07-fold in experiment 1 and 2 respectively. The test material is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations ≥ 1000 μM.

Under the conditions of this study, the test material is classified as negative as it showed no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 November 2017 to 08 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 442E – Annex II ‘In Vitro Skin Sensitisation: U937 Cell Line Activation Test (USENS ™)’

Version / remarks:

9 October 2017

Deviations:

no

GLP compliance:

yes

Type of study:

other: U937 Cell Line Activation Test

Justification for non-LLNA method:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended for skin sensitisation to minimize the need of in vivo testing. One of the validated in vitro skin sensitisation tests is the U-SENSTM assay, which is recommended in international guideline (e.g. OECD).

Specific details on test material used for the study:

- No correction was made for the composition/purity of the test material.
- A solubility test was performed. The test material was either dissolved or suspended in complete medium and DMSO to a final concentration of 50 mg/mL. The test material formed a clear solution in complete medium at 50 mg/mL (yellow solution). In DMSO the test material formed homogenous suspension at 50 mg/mL (white suspension). Complete medium was used as solvent for the main experiments.
- In the main experiments the test material was dissolved in complete medium at 0.4 mg/mL. The stock was diluted to a final test concentrations of 200, 100, 50, 20, 10 and 1.0 μg/mL and 200, 180, 140 and 100 μg/mL in the 96-well plate in experiment 1 and 2, respectively.
- No precipitation was observed at the end of the incubation period in the 96-well plates.
- Test material concentrations were used within 3.5 hours after preparation.
- Any residual volumes were discarded.

Details on the study design:

TEST SYSTEM
- Test System: U937 human monocytes.
- Justification: Inducible CD86-expressing cells
- Source: ATCC (American Type Culture Collection, Virginia, USA). ATCC no.: CRL-1593.2TM.
- Stock cultures of these cells are stored in liquid nitrogen (-196 °C). Cells were used after an acclimatisation period of approximately 8 days after thawing and were not sub-cultured more than 21 times. Once a year the cell line is checked for infection with a mycoplasma detection test.
- Each batch of cells received from a supplier are submitted to a qualification process to guarantee their suitability (spontaneous CD86 level) for the test by comparison with the historical data or data from the literature.
- Cell culture:
Cell culture medium: Stock and treatment cultures were performed in RPMI-1640 medium supplemented with 10 % (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (FCS), L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively).
Environmental conditions: All incubations were carried out in a humid atmosphere of 80 – 100 % (actual range 90 – 101 %) containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 36.3 –36.6 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

EXPERIMENTAL DESIGN
- Plating of Cells: Cultures were initiated in 96-well plates using 100 μL/well of a cell suspension adjusted at 5.0 x 10^5 viable cells/mL. If the cell viability is < 90 % the cells were not used. All assays were performed using two replicate culture-wells for the test material. One replicate was dedicated to the nonspecific IgG1 binding and the other one to the CD86 binding. Three replicates of complete medium untreated control, solvent/vehicle control, negative and positive controls were tested.
- Number of experiments: Two valid experiments were conducted per test material to demonstrate reproducibility of the results and conclusion. Initially, experiment 1 did not pass all the acceptability criteria and therefore this part of the study was repeated.
- Treatment of Cells: Cells are treated for 45 ± 3 hours with the selected doses. The test material was in the first experiment evaluated up to 200 μg/mL using six doses: 1.0, 10, 20, 50, 100 and 200 μg/mL. A negative untreated control (culture medium), a vehicle control and the positive and negative control materials were included. The final volume in the wells was 200 μL. In the second experiment cells were treated with four selected doses of test material. At least 2 concentrations were common with the previous experiment. The concentrations selected in the second experiment were 100, 140, 180 and 200 μg/mL.
- Precipitate evaluation: After 45 ± 3 hours of exposure, wells were checked for precipitate.
- Cell antibodies staining for IgG1 and CD86: Cultures were transferred into V-shaped 96-well plates. The cells were separated from the exposure medium by centrifugation (5 min, 200 g). The supernatant was discarded and cells were rinsed once with Phosphate Buffered Saline (PBS) containing 5 % FCS. After a second centrifugation step 100 μL/well of staining buffer (PBS containing 5 % FCS) was applied to the cells. FITC-conjugated antibodies was used for both IgG1 and CD86 staining: Mouse IgG1 of unknown specificity, for isotypic control and Human CD86 specific mouse IgG1. The cells were transferred into new V-shaped 96-well plates (keeping the same plate template) containing 5 μL/well of the appropriate antibody (1:1 diluted in PBS) and placed refrigerated in the dark for 30 minutes. After this staining period, the cells were rinsed twice with a mixture of PBS/FCS and once in PBS alone and re-suspended in 90 μL of PBS.

- Flow cytometry method:
Acquisition: Just before acquisition, 5 μL of a 0.5 μg/mL propidium iodide (PI) solution was added to each well. The size (FSC) was set linear and the granularity (SSC) parameter was set to logarithmic scale and a R1 region was defined in which approximately 10,000 events were acquired for each culture. The acquisition parameters remained unchanged for the acquisition of all the wells. When the cell viability was low, up to 20,000 cells including dead cells could be acquired. Alternatively, data can be acquired for one minute after the initiation of the analysis. For the acquisition the BD FACSCanto™ flow cytometer was used and for further analysis BD FACSDiva™ software was used.
Analysis: All analysis parameters were set on the RPMI wells for IgG1 and remained unchanged, for the analysis of all the other wells. The P1 region was adjusted if necessary in a SSC (X-axis) and FSC (Y-axis) plot. The P2 region was defined for the PI negative cells among P1 in a histogram with counts (Yaxis) and PI fluorescence (X-axis). The amount of cytotoxicity were analysed as percentage of cells in P2. The P2 region was then plotted in a Dot-plot as fluorescence (X-axis) and SSC (Y-axis) and a quadrant was placed according acceptability criterion b. The percentage of cells in the UR quadrant was used to calculate the stimulation index.
Colour Interferences: There is colour interference in the IgG1 evaluation when the X Median of the FITCfluorescence in the UL Quad is 50 % higher than the X Median fluorescence of the vehicle control IgG1 well (IgG1 X Median S.I. ≥ 150 %).

ACCEPTABILITY CRITERIA
- At the end of the incubation treatment period, the mean viability of the triplicate untreated U937 cells is > 90 %
- When DMSO is used as a solvent, the validity of the DMSO vehicle control is assessed by calculating a DMSO S.I. compared to untreated cells, and the mean viability of the triplicate cells is > 90 %. The DMSO vehicle control is valid if the mean value of its triplicate CD86 S.I. was smaller than 250 % of the mean of the triplicate CD86 S.I. of untreated U937 cells.
- The CD86 basal expression of untreated U937 cells is within the range of ≥ 2 % and ≤ 25 %.
- At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6 % and < 1.5 %.
- No drift in CD86 expression is observed. A drift is defined by i) the corrected %CD86+ value of the untreated control replicate 3 is less than 50 % of the mean of the corrected %CD86+ value of untreated control replicates 1 and 2; and ii) the corrected %CD86+ value of the negative control replicate 3 is less than 50 % of mean of the corrected %CD86+ value of negative control replicates 1 and 2.
- The positive control (TNBS) is considered as valid if at least two out of the three wells are positive (CD86 S.I. ≥ 150 %) and non-cytotoxic (cell viability ≥ 70 %).
- Negative control LA is considered as valid if at least 2 out of 3 LA wells are negative (CD86-IgG1 SI < 150%) and non-cytotoxic (cell viability ≥ 70 %).
If (one of) the acceptability criteria are not met and the Study Director decides that this has a critical effect on the study, the test was rejected and repeated.

ANALYSIS
- CV70 calculations: When the CV70 cannot be calculated it is indicated as “NA” (Not Applicable). Otherwise the calculation is done as follows: For each culture (IgG1 well and CD86 well), the percentage of viable cells (PI negative cells) was evaluated. The viability for each dose level is the mean of the IgG1 well and CD86 well. The theoretical concentration at which the chemical induces 30 % cytotoxicity (i.e., 70 % viability) was calculated using the following formula:
CV70 = C1+ [ ((V1 – 70) / (V1 – V2)) x (C2 – C1)]
Where:
V1 = the first percentage of viability above 70 %
V2= the first percentage of viability below 70 %
C1 = dose level corresponding to V1
C2 = dose level corresponding to V2
- EC150 calculations: When the EC150 cannot be calculated it is indicated as “NA” (Not Applicable). Otherwise the calculation were done as follows: For each CD86 well culture, the percentage of induced CD86+ cells is calculated as: [absolute %CD86+ — absolute%IgG1+]
A stimulation index (S.I.) is calculated for each dose level as follows:
S.I. = [([%CD86+ - %IgG1+] in the treated culture / Mean [%CD86+ - %IgG1+] of the vehicle cultures) x 100
The viability for each dose level are the mean of the IgG1 well and CD86 well. The theoretical concentration at which the chemical induces a S.I. of 150 (i.e., 50 % of CD86+ cells over the vehicle control) was calculated using the following formula:
EC150 = C1 + [ (150 – S.I. 1)/ (S.I. 2 – S.I. 1)] x (C2 – C1)
Where:
S.I.1 = the first percentage of CD86+ below 150 %
S.I.2 = the first percentage of CD86+ above 150 %
C1 = dose level corresponding to S.I. 1
C2 = dose level corresponding to S.I. 2

INTERPRETATION
- For CD86 expression measurement, each test chemical is tested in at least two independent runs (performed on a different day) to derive a single prediction (NEGATIVE or POSITIVE).
- The individual conclusion of an U-SENS™ run is considered Negative (hereinafter referred to as N) if the S.I. of CD86 is less than 150 % at all non-cytotoxic concentrations (cell viability ≥ 70 %) and if no interference (cytotoxicity, solubility or colour) is observed. Solubility interference is defined as crystals or drops observed under the microscope at 45 ± 3h post treatment (before the cell staining). Colour interference is defined as a shift of the FITC-labelled IgG1 dot-plot (IgG1 FL1 Geo Mean S.I. ≥ 150 %).
- In all other cases: S.I. of CD86 higher or equal to 150 % and/or interferences observed, the individual conclusion of an U-SENS™ run is considered Positive (hereinafter referred to as P).
- An U-SENS™ prediction is considered NEGATIVE if at least two independent runs are negative (N). If the first two runs are both negative (N), the U-SENS™ prediction is considered NEGATIVE and a third run does not need to be conducted.
An U-SENS™ prediction is considered POSITIVE if at least two independent runs are positive (P). If the first two runs are both positive (P), the U-SENS™ prediction is considered POSITIVE and a third run does not need to be conducted.
- There is an exception if, in the first run, the S.I. of CD86 is higher or equal to 150 % at the highest non-cytotoxic concentration only. The run is then concluded NO CONCLUSION (NC), and additional concentrations (between the highest non cytotoxicity concentration and the lowest cytotoxicity concentration) should be tested in additional runs. A run NC conducts automatically to the need of at least 2 more runs, and to a fourth run in case runs 2 and 3 are not concordant (N and/or P independently). Follow up runs will be considered positive even if only one non cytotoxic concentration gives a CD86 equal or above 150 %, since the dose setting has been adjusted for the specific test chemical. The final prediction will be based on the majority result of the three or four individual runs (i.e. 2 out of 3 or 2 out of 4).

Positive control results:

Experiment 1: The positive control (TNBS) showed a S.I. ≥ 364 % in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).
Experiment 2: The positive control (TNBS) showed a S.I. ≥ 426 % in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

Key result

Run / experiment:

other: Experiments 1 and 2

Parameter:

other: CV70 (µg/mL)

Value:

200

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The viability of the cells was higher than 70 % at all test concentrations and therefore no CV70 values could be calculated and is considered to be higher than 200 μg/mL.

Key result

Run / experiment:

other: Experiments 1 and 2

Parameter:

other: EC150 (µg/mL)

Value:

2 000

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No increase in expression levels of CD86 compared to the vehicle control was observed at any of the test concentrations after treatment with the test material. No EC150 could be calculated and is considered to be higher than 200 μg/mL.

Other effects / acceptance of results:

EXPERIMENT 1
- No precipitation was observed at the end of the incubation period in the 96-well plates.
- The test material showed no toxicity, the viability of the cells was higher than 70 % at all test concentrations and therefore no CV70 values could be calculated and is considered to be higher than 200 μg/mL.
- No increase in expression levels of CD86 compared to the vehicle control was observed at any of the test concentrations after treatment with the test material. No EC150 could be calculated and is considered to be higher than 200 μg/mL.
- The test material showed no colour interference.
- The positive control (TNBS) showed a S.I. ≥ 364 % in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a S.I. < 90 % in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70 %).

EXPERIMENT 2
- No precipitation was observed at the end of the incubation period in the 96-well plates.
- The test material showed no toxicity, the viability of the cells was higher than 70 % at all test concentrations and therefore no CV70 values could be calculated and is considered to be higher than 200 μg/mL.
- No increase in expression levels of CD86 compared to the vehicle control was observed at any of the test concentrations after treatment with the test material. No EC150 could be calculated and is considered to be higher than 200 μg/mL.
- The test material showed no colour interference.
- The positive control (TNBS) showed a S.I. ≥ 426 % in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a S.I. < 101 % in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70 %).

ACCEPTABILITY CRITERIA
- At the end of the incubation treatment period, the mean viability of the triplicate untreated U937 cells was above the threshold of 90 % (97 % in experiment 1 and 96 % in experiment 2).
-The CD86 basal expression of untreated U937 cells is within the range of ≥ 2 % and ≤ 25 % in both experiments.
- At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6 % and < 1.5 % in both experiments.
- No drift in CD86 expression was observed in the untreated controls and negative controls.
In both experiment the positive and negative control were considered valid. Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

DISCUSSION
The test material showed no toxicity (CV70 value >200 μg/mL) and no biologically relevant induction of the CD86 activity (EC150 value >200 μg/mL) was measured at any of the test concentrations in both experiments. The test material is classified as negative in the U-Sens™ assay since negative results (< 150 % increase) were observed at all test concentrations with a cell viability of >70 % compared to the vehicle control.

Table 1: Overview Stimulation index of CD86 and Cell Viability in Experiment 1 and 2 of The Test Material

Dose Level (µg/mL)	% Viability (mean)		CD86-IgG1 S.I.		Colour Interference S.I.
	Experiment 1	Experiment 2	Experiment 1	Experiment 2	Experiment 1	Experiment 2
1	97	-	96	-	108	-
10	96	-	97	-	106	-
20	96	-	95	-	109	-
50	97	-	103	-	102	-
100	97	96	90	96	109	108
140	-	96	-	88	-	115
180	-	95	-	96	-	110
200	96	96	123	111	113	109

Interpretation of results:

other: Negative in the U-Sens™ assay

Conclusions:

Under the conditions of this study, the test material is classified as negative (no increase in the expression levels of CD86 cell surface marker in the U937 cell line).

Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 442E, under GLP conditions.

The objective of this study was to evaluate the ability of the test material to increase the expression levels of CD86 cell surface marker in the U937 cell line in the U937 cell line activation Test (U-Sens™) assay.

The test material was dissolved in complete medium at 0.4 mg/mL. In the first experiment the stock was diluted to six test concentration (1, 10, 20, 50, 100 and 200 μg/mL). In the second experiment, a more narrow dose-response analysis was performed. No precipitate was observed at any dose level tested. Two independent experiments were performed.

Both experiments passed the acceptance criteria: At the end of the incubation treatment period, the mean viability of the triplicate untreated U937 cells was above the threshold of 90 % (97 % in experiment 1 and 96 % in experiment 2); The CD86 basal expression of untreated U937 cells is within the range of ≥ 2 % and ≤ 25 % in both experiments; At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6 % and < 1.5 % in both experiments; No drift in CD86 expression was observed in the untreated controls and negative controls. In both experiment the positive and negative control were considered valid. Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

The test material showed no toxicity (CV70 value >200 μg/mL) and no biologically relevant induction of the CD86 activity (EC150 value >200 μg/mL) was measured at any of the test concentrations in both experiments. The test material is classified as negative in the U-Sens™ assay since negative results (< 150 % increase) were observed at all test concentrations with a cell viability of >70 % compared to the vehicle control.

Under the conditions of this study, the test material is classified as negative (no increase in the expression levels of CD86 cell surface marker in the U937 cell line).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

DPRA, Reinen (2017)

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 442C, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of this study was to determine the reactivity of the test material towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test material with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test material to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

Milli-Q water (MQ) was found to be an appropriate solvent to dissolve the test material and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study.

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A, C and Cwater, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test material, were all within the acceptability criteria for the DPRA.

In the cysteine reactivity assay the test material showed 7.0 % SPCC depletion while in the lysine reactivity assay the test material showed 5.9 % SPCL depletion. The mean of the SPCC and SPCL depletion was 6.5 % and as a result the test material was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Under the conditions of this study the test material was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Keratinosens, Westerink (2017)

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD TG 442D, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of this study was to evaluate the ability of the test material to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay.

The test material was suspended in dimethyl sulfoxide at 200 mM. From this stock 11 spike solutions in dimethyl sulfoxide were prepared. At concentrations of 50 mM and higher the test material formed a suspension in dimethyl sulfoxide whereas at 25 mM and lower it was fully soluble. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 μM (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. No precipitate was observed at any dose level tested. Two independent experiments were performed.

Both experiments passed the acceptance criteria: 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 (107 μM and 29 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.56-fold and 3.48-fold in experiment 1 and 2, respectively); Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20 % (8.2 and 9.9 % in experiment 1 and 2, respectively). Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

The test material showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.05-fold and 1.07-fold in experiment 1 and 2 respectively. The test material is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations ≥ 1000 μM.

Under the conditions of this study, the test material is classified as negative as it showed no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes.

U-SENS, Eurlings (2018)

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 442E, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of this study was to evaluate the ability of the test material to increase the expression levels of CD86 cell surface marker in the U937 cell line in the U937 cell line activation Test (U-Sens™) assay.

The test material was dissolved in complete medium at 0.4 mg/mL. In the first experiment the stock was diluted to six test concentration (1, 10, 20, 50, 100 and 200 μg/mL). In the second experiment, a more narrow dose-response analysis was performed. No precipitate was observed at any dose level tested. Two independent experiments were performed.

Both experiments passed the acceptance criteria: At the end of the incubation treatment period, the mean viability of the triplicate untreated U937 cells was above the threshold of 90 % (97 % in experiment 1 and 96 % in experiment 2); The CD86 basal expression of untreated U937 cells is within the range of ≥ 2 % and ≤ 25 % in both experiments; At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6 % and < 1.5 % in both experiments; No drift in CD86 expression was observed in the untreated controls and negative controls. In both experiment the positive and negative control were considered valid. Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

The test material showed no toxicity (CV70 value >200 μg/mL) and no biologically relevant induction of the CD86 activity (EC150 value >200 μg/mL) was measured at any of the test concentrations in both experiments. The test material is classified as negative in the U-Sens™ assay since negative results (< 150 % increase) were observed at all test concentrations with a cell viability of >70 % compared to the vehicle control.

Under the conditions of this study, the test material is classified as negative (no increase in the expression levels of CD86 cell surface marker in the U937 cell line).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

To conclude on the skin sensitisation potential of the test material, it is taken into account that two out of three in-vitro studies on skin sensitisation showed a clear negative result. In the DPRA, the test material was classified in the “low reactivity class” and the mean peptide depletion was just above the threshold. Moreover, in-silico prediction resulted in having no alerts for skin sensitisation.

In a weight-of-evidence approach it can be concluded that the test material is not sensitising to human skin and therefore, it has not to be classified.