4-chloromethyl-4''-ethyl-2'-fluoro-[1,1':4',1''-terphenyl]

Endpoint summary

Description of key information

Due to the observed precipitation in the cysteine peptide experiment a prediction cannot be made for the test item in an OECD TG 442 C study (reference 7.4.1-1).

The test item did induce the luciferase activity in a study according to OECD TG 442D and thus, the test item can be considered as sensitiser (reference 7.4.1-2).

The test item did upregulate the cell surface marker CD86 in at two independent experiment runs in an OECD TG 442E study. Therefore, the test item is considered to be a skin sensitiser (reference 7.4.1-3).

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:

weight of evidence

Study period:

2017-12-01 to 2018-01-20

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:

February 04, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions: 20.32 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (39.48 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM. 20.56 mg lysine peptide with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (39.09 mL) to reach a concentration of 0.667 mM. All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.
- Preparation of the test chemical solutions: The test item was freshly prepared immediately prior to use. The test item was pre-weighed into a glass vial and was dissolved in an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared. In a pre-experiment the test item was not soluble in acetonitrile, dist. water, dist. water : acetonitrile 1:1 (v/v), isopropanol, methanol, 1,4-butanediol. The test item was completely soluble in N,N-dimethylformamide(DMF), therefore, DMF was chosen as suitable vehicle for the main experiments.
- Preparation of the positive controls, reference controls and co-elution controls:
Positive Control: Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100mM was prepared and was included in every assay run for both peptides.
Co-elution Control: Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides.
Reference Control: Reference controls (RCs) were set up in parallel to sample preparation in order to verify the validity of the test run. Reference control A was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run. Reference control B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each HPLC run. Reference control C was set up for the test item and the positive control. RC C for the positive control was prepared using acetonitrile. RC C for the test item was prepared using the respective solvent used to solubilise the test item. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples.

INCUBATION
- Incubation conditions: The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. Reference controls, co-elution controls as well as the positive control were set up in parallel. Samples were prepared according to the scheme described in table 1 (see “Any other information on materials and methods”).
- Precipitation noted:
For the 100 mM solution of the test item precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged prior to the HPLC analysis.
For the 100 mM stock solution of the test item precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged prior to the HPLC analysis. Phase separation (droplets) was observed for the co-elution control of the positive control. The sample was not centrifuged prior to the HPLC analysis.

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys: A standard calibration curve was generated for both, the cysteine and the lysine peptide. Peptide standards were prepared in a solution of 20 % acetonitrile : 80 % buffer ( v / v ) using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide (dilution buffer (DB)). A serial dilution of the peptide stock solution (0.667 mM) using the respective DB was performed, resulting in 7 calibration solutions in the range from 0.0 to 0.534 mM.
- Verification of the suitability of the HPLC for test chemical and control substances: Peptide depletion was monitored by HPLC coupled with an UV detector at λ = 220 nm using a reversed-phase HPLC column (Zorbax SB-C-18 2.1 mm x 100 mm x 3.5 micron) as preferred column. The entire system was equilibrated at 30 °C with 50 % phase A and 50 % phase B for at least 2 hours before running the analysis sequence. The HPLC analysis was performed using a flow rate of 0.35 mL/min and a linear gradient from 10 % to 25 % acetonitrile over 10 minutes, followed by a rapid increase to 90 % acetonitrile. The column was re-equilibrated under initial conditions for 7 minutes between injections. Equal volumes of each standard, sample and control were injected. HPLC analysis for the cysteine and lysine peptide was performed concurrently (if two HPLC systems were available) or on separate days. If analysis was conducted on separate days all test chemical solutions were freshly prepared for both assays on each day. The analysis was timed to assure that the injection of the first sample started 22 to 26 hours after the test chemical was mixed with the peptide solution. The HPLC run sequence was set up in order to keep the HPLC analysis time less than 30 hours. For details on HPLC system refer to “Any other information on materials and methods”.

DATA EVALUATION
- Cys and Lys peptide detection wavelength: λ = 220 nm

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 62.40 %.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

5.58

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

0.21

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

other: Due to the observed precipitation in the cysteine peptide experiment a prediction cannot be made.

Other effects / acceptance of results:

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

DEMONSTRATION OF TECHNICAL PROFICIENCY: not specified

ACCEPTANCE OF RESULTS:
Please refer to table 8 and 9 under “Any other information on results”.

 

Table 2: Cysteine and Lysine Values of the Calibration Curve

Sample	Cysteine Peptide		Lysine Peptide
	Peak Area at 220 nm	Peptide Concentration [mM]	Peak Area at 220 nm	Peptide Concentration [mM]
STD1	5221.6792	0.5340	4254.6284	0.5340
STD2	2648.5273	0.2670	2161.7087	0.2670
STD3	1327.5009	0.1335	1051.8925	0.1335
STD4	670.7400	0.0667	525.2503	0.0667
STD5	334.8562	0.0334	260.5259	0.0334
STD6	166.3505	0.0167	130.8325	0.0167
STD7	0.0000	0.0000	0.0000	0.0000

 

Table 3: Depletion of the Cysteine Peptide

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1609.4324	0.1633	68.11	68.17	0.05	0.08
	1604.4860	0.1628	68.21
	1605.4248	0.1629	68.19
Test Item	4722.0444	0.4814	4.97	5.58	0.54	9.77
	4682.2471	0.4774	5.77
	4670.3384	0.4761	6.01

 

Table 4: Depletion of the Lysine Peptide

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1675.9452	0.2100	57.93	56.62	1.13	2.00
	1753.5808	0.2197	55.98
	1754.5223	0.2198	55.95
Test Item	4024.7078	0.5039	0.13	0.21	0.15	72.44
	4025.2893	0.5039	0.12
	4014.3962	0.5026	0.39

 

Table 5: Prediction Model 1

Cysteine 1:10/ Lysine 1:50 Prediction Model ¹

Mean Cysteine andLysine PPD	Reactivity Class	DPRA Prediction²
0.00% ≤ PPD ≤ 6.38%	No or Minimal Reactivity	Negative
6.38% < PPD ≤ 22.62%	Low Reactivity	Positive
22.62% < PPD ≤ 42.47%	Moderate Reactivity
42.47% < PPD ≤ 100%	High Reactivity

1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2 DPRA predictions should be considered in the framework of an IATA.

 

 

Table 6: Prediction Model 2

Cysteine 1:10 Prediction Model

Cysteine PPD	ReactivityClass	DPRA Prediction²
0.00% ≤ PPD ≤ 13.89%	No or Minimal Reactivity	Negative
13.89% < PPD ≤ 23.09%	Low Reactivity	Positive
23.09% < PPD ≤ 98.24%	Moderate Reactivity
98.24% < PPD ≤ 100%	High Reactivity

 

Table 7: Categorization of the Test Item

Prediction Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)			Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	-	-	-	5.58	Minimal Reactivity	negative
Positive Control	62.40	High Reactivity	positive	68.17	Moderate Reactivity	positive

 

Table 8: Acceptance Criteria for Cysteine Peptide

Cysteine Peptide Run
Acceptance Criterion	Range	Value	pass/fail
coefficient of determination	R² >0.99	0.9999	pass
mean peptide concentration of RC A	0.45 ≤ x ≤ 0.55 mM	0.5193	pass
mean peptide concentration of RC C (PC)	0.45 ≤ x ≤ 0.55 mM	0.5147	pass
mean peptide concentration of RC C (TI)	0.45 ≤ x ≤ 0.55 mM	0.5066	pass
CV of the peak area of RC B	<15 %	0.44	pass
CV of the peak area of RC C (PC)	<15 %	0.53	pass
CV of the peak area of RC C (TI)	<15 %	0.34	pass
mean peptide depletion of the PC	60.8 % < x < 100 %	68.17	pass
SD of peptide depletion of the PC replicates	<14.9 %	0.05	pass
SD of peptide depletion of the TI replicates	<14.9 %	0.54	pass

 

Table 9: Acceptance Criteria for Lysine Peptide

Lysine Peptide Run
Acceptance Criterion	Range	Value	pass/fail
coefficient of determination	R² >0.99	0.9999	pass
mean peptide concentration of RC A	0.45 ≤ x ≤ 0.55 mM	0.5003	pass
mean peptide concentration of RC C (PC)	0.45 ≤ x ≤ 0.55 mM	0.4987	pass
mean peptide concentration of RC C (TI)	0.45 ≤ x ≤ 0.55 mM	0.5045	pass
CV of the peak area of RC B	<15 %	0.48	pass
CV of the peak area of RC C (PC)	<15 %	0.47	pass
CV of the peak area of RC C (TI)	<15 %	0.32	pass
mean peptide depletion of the PC	40.2 % < x < 69.0 %	56.62	pass
SD of peptide depletion of the PC replicates	<11.6 %	1.13	pass
SD of peptide depletion of the TI replicates	<11.6 %	0.15	pass

 

Interpretation of results:

study cannot be used for classification

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards the cysteine peptide. Due to the observed precipitation in the cysteine peptide experiment a prediction cannot be made.

Executive summary:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

In the present study, according to OECD TG 442C, the test item was dissolved in N.N-dimethylformamide (DMF) based on the results of the pre-experiments. Based on a molecular weight of 324.82 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM solution of the test item precipitation was observed when diluted with the cysteine peptide solution. After the 24 h± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged prior to the HPLC analysis.

For the 100 mM stock solution of the test item precipitation was observed when diluted with the lysine peptide solution. After the 24 h± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged prior to the HPLC analysis. Phase separation (droplets) was observed for the co-elution control of the positive control. The sample was not centrifuged prior to the HPLC analysis.

A minor co-elution of the test item with the lysine peptide peak was observed. The peak area determined in the co-elution controls corresponds to 0.0104 mM of lysine peptide. Therefore, the given peak areas and corresponding lysine peptide values can only be considered as an estimation of the peptide depletion and cannot be used for evaluation.

The 100 mM stock solution of the test item showed minimal reactivity towards the cysteine peptide. The mean depletion of the cysteine peptide was ≤ 13.89 % (5.58 %). According to the evaluation criteria in the guideline, if a precipitation or phase separation is observed after the incubation period, peptide depletion may be underestimated and a conclusion on the lack of reactivity cannot be drawn with sufficient confidence in case of a negative result. Due to the observed precipitation in the cysteine experiment no prediction can be made.

Since precipitation was observed for the lysine and cysteine peptide samples containing also test item after the incubation period, no final prediction can be made.

In this study under the given conditions the test item showed minimal reactivity towards the cysteine peptide. Due to the observed precipitation in the cysteine peptide experiment a prediction cannot be made.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-11-14 to 2018-02-23

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 04, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: All test item solutions were freshly prepared immediately prior to use. The test item was dissolved in Tetrahydrofuran (THF, purity ≥99 %; Merck; Lot No.: I864407, Sigma, Lot No.: STBH3251). A stock solution of 200 mM was prepared by pre-weighing the test material into a glass vial. A stable suspension was formed when diluted 1:100 in cell culture medium.
- Preparation of the test chemical serial dilutions: Based on the stock solution a set of twelve master solutions in 100 % solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a 4 % share of the solvent. Since the test item was dissolved in THF, DMSO was added at a final concentration of 1 % (v/v). These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1 % (v/v) in all test item concentrations and controls.
- Preparation of the positive controls: Cinnamic aldehyde (CA, (2E)-3-phenylprop-2-enal; CAS 104-55-2; >98 %; Sigma; Lot No.: MKBS2662V, Alfa Aesar; Lot No.: 10176010) was used as positive control. CA was dissolved in DMSO (AppliChem; Lot No.: 0000978834, 001055932) at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM - 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 µM – 64 µM. The final concentration of the solvent DMSO was 1 % (v/v) for all wells. Furthermore, 1 % (v/v) of THF was added to the positive control.
- Preparation of the solvent, vehicle and negative controls:
Blank: A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.
Negative Control: DMSO (AppliChem; Lot No.: 0000978834, 01001055932) at a final concentration of 1 % (v/v) in test item exposure medium was used as negative control. Furthermore, 1 % (v/v) of THF was added to the negative control. Six wells were included in every testing plate. The preparation of the negative control was carried out analogous to the test item.
- Stable dispersion obtained: Stable suspension was formed in cell culture medium.

DOSE RANGE FINDING ASSAY: No

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: test item: 3, positive control: 3, negative control: 6/plate
- Number of repetitions: 2
- Test chemical concentrations: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM
- Application procedure: A cell suspension of 8 × 104 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1 × 104 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability. After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5 % CO2. Thereafter, the assay medium was discarded and replaced by 150 µL test item exposure medium. 50 µL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item. All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross- contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5 % CO2.
- Exposure time: 48 h ± 1 h
- Study evaluation and decision criteria used: For each test item two independent repetitions using separately prepared test item solutions and independently harvested cells are necessary to derive a prediction. Each independent run consisted of three replicates for every concentration step of the test item and the positive control. In case of discordant results a third independent run is performed. For every concentration showing >1.5 fold luciferase activity induction, statistical significance (p <0.05) was calculated using a two-tailed Student’s t-test comparing the luminescence values for the three replicated samples with the luminescence values in the solvent (negative) control wells. The lowest concentration with >1.5 fold luciferase activity induction was the value determining the EC1.5 value. It was checked in each case whether this value was below the IC30 value, indicating that there was less than 30 % reduction on cellular viability at the EC1.5 determining concentration.
- Description on study acceptance criteria: The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20 % in each repetition.

SEEDING AND INCUBATION
- Seeding conditions: passage number <25 (P 6 in experiment 1; P 3 in experiment 2), density see above (“Application procedure”)
- Incubation conditions: 37  1°C and 5 % CO2
- Washing conditions: once with DPBS
- Precipitation noted: No

LUCIFERASE ACTIVITY MEASUREMENTS
- Choice of luminometer with demonstration of appropriate luminescence measurements based on control test: not specified
- Plate used: not specified
- Lysate preparation: 20 µL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light.

DATA EVALUATION
- Cytotoxicity assessment: For the cell viability plate the medium was replaced with 200 µL test item exposure medium. 27 µL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5 % CO2. Afterwards the medium was removed and replaced by 200 µL 10 % SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5 % CO2 overnight (experiment 1) and over the weekend (experiment 2). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.
- Prediction model used: The test item is considered positive in accordance with UN GHS “Category 1” if the following conditions were met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70 % at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is <1000 µM
- an apparent overall dose-response for luciferase induction
If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <1000 µM is considered as inconclusive. A negative result for test items with a log KOW > 7 has to be interpreted with care due to the applicability of the test method.

Vehicle / solvent control:

other: 1 % (v/v) DMSO and 1 % (v/v) THF in test item exposure medium

Negative control:

not applicable

Positive control:

cinnamic aldehyde [442D]

Positive control results:

The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (7.59(experiment 1); 3.43 (experiment 2)) and thus, met the acceptability criteria.

Group:

test chemical

Run / experiment:

mean

Parameter:

IC50 [442D]

Value:

11.84

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

mean

Parameter:

IC30 [442D]

Value:

6.71

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

6.41

Cell viability:

29.4 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

3.23

Cell viability:

40.6 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

not determinable

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

not determinable

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

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

DEMONSTRATION OF TECHNICAL PROFICIENCY: not specified

ACCEPTANCE OF RESULTS:
Please refer to “Any other information on results”.

Results of the Cytotoxicity Measurement

	Concentration [µM]	Cell Viability [%]
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	100	100	100	0.0
Positive Control	4.00	87.6	83.5	85.6	2.8
	8.00	92.1	87.0	89.6	3.6
	16.00	100.1	96.3	98.2	2.7
	32.00	106.4	100.6	103.5	4.1
	64.00	112.2	111.7	111.9	0.4
Test Item	0.98	133.1	114.0	123.5	13.5
	1.95	126.1	119.8	122.9	4.4
	3.91	97.6	108.9	103.2	8.0
	7.81	50.8	63.0	56.9	8.7
	15.63	42.7	49.1	45.9	4.5
	31.25	29.4	40.6	35.0	7.9
	62.50	29.1	37.5	33.3	5.9
	125.00	27.1	40.0	33.5	9.1
	250.00	29.4	45.8	37.6	11.6
	500.00	35.7	47.4	41.5	8.2
	1000.00	39.3	57.0	48.2	12.6
	2000.00	59.0	64.8	61.9	4.1

 

Induction of Luciferase Activity Experiment 1

Experiment 1	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.24	1.44	1.13	1.27	0.16
	8.00	1.21	1.45	1.24	1.30	0.13
	16.00	1.49	1.44	1.46	1.46	0.02
	32.00	2.30	1.82	2.32	2.15	0.28	*
	64.00	5.99	8.93	7.86	7.59	1.49	*
Test Item	0.98	1.96	2.54	1.85	2.12	0.37	*
	1.95	3.31	2.50	2.06	2.62	0.64	*
	3.91	3.58	3.81	3.48	3.63	0.17	*
	7.81	5.86	5.45	4.93	5.41	0.46	*
	15.63	3.64	3.82	3.02	3.49	0.42	*
	31.25	7.10	6.91	5.23	6.41	1.03	*
	62.50	6.17	5.77	5.03	5.66	0.58	*
	125.00	4.87	5.34	4.79	5.00	0.30	*
	250.00	4.09	5.31	4.29	4.56	0.66	*
	500.00	4.21	4.13	3.96	4.10	0.13	*
	1000.00	3.30	4.34	3.28	3.64	0.60	*
	2000.00	2.29	3.47	2.34	2.70	0.67	*

* = significant induction according to Student’s t-test, p<0.05

 

Induction of Luciferase Activity Experiment 2

Experiment 2	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.05	1.07	1.12	1.08	0.04
	8.00	1.17	1.29	1.25	1.24	0.07
	16.00	1.31	1.35	1.68	1.45	0.20
	32.00	1.93	1.85	1.69	1.83	0.12	*
	64.00	3.67	2.88	3.74	3.43	0.48	*
Test Item	0.98	1.47	1.74	1.54	1.58	0.14	*
	1.95	2.16	2.65	2.55	2.45	0.26	*
	3.91	2.42	2.74	2.39	2.52	0.20	*
	7.81	2.80	3.41	2.73	2.98	0.37	*
	15.63	2.61	3.35	3.25	3.07	0.40	*
	31.25	3.09	3.58	3.03	3.23	0.30	*
	62.50	2.85	3.33	3.20	3.13	0.25	*
	125.00	3.07	3.13	2.92	3.04	0.11	*
	250.00	3.14	3.07	2.94	3.05	0.10	*
	500.00	3.09	2.73	2.77	2.86	0.20	*
	1000.00	2.88	2.37	2.83	2.69	0.28	*
	2000.00	2.39	2.21	2.43	2.34	0.12	*

* = significant induction according to Student’s t-test, p<0.05

 

Induction of Luciferase Activity – Overall Induction

Overall Induction	Concentration [µM]	Fold Induction				Significance
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	1.00	1.00	1.00	0.00
Positive Control	4.00	1.27	1.08	1.18	0.13
	8.00	1.30	1.24	1.27	0.04
	16.00	1.46	1.45	1.46	0.01
	32.00	2.15	1.83	1.99	0.23	*
	64.00	7.59	3.43	5.51	2.94
Test Item	0.98	2.12	1.58	1.85	0.38
	1.95	2.62	2.45	2.54	0.12	*
	3.91	3.63	2.52	3.07	0.78
	7.81	5.41	2.98	4.20	1.72
	15.63	3.49	3.07	3.28	0.30	*
	31.25	6.41	3.23	4.82	2.25
	62.50	5.66	3.13	4.39	1.79
	125.00	5.00	3.04	4.02	1.39
	250.00	4.56	3.05	3.81	1.07
	500.00	4.10	2.86	3.48	0.87
	1000.00	3.64	2.69	3.17	0.67	*
	2000.00	2.70	2.34	2.52	0.25	*

 * = significant induction according to Student’s t-test, p<0.05

 

Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Solvent Control	< 20 %	9.3	pass	9.1	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.0	pass	2.0	pass
EC1.5 PC	7 < x < 34 µM	16.83	pass	18.27	pass
Induction PC at 64 µM	2.00 < x < 8.00	7.59	pass	3.43	pass

 

Historical Data

Acceptance Criterion	Range	Mean	SD	N
CV Solvent Control	< 20 %	11.3	3.3	41
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.3	0.6	41
EC1.5 PC	7 < x < 34 µM	20.4	6.7	41
Induction PC at 64 µM	2.00 < x < 8.00	3.3	1.1	41

 

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test item did induce the luciferase activity and thus, the test item can be considered as sensitiser.

Executive summary:

 

In the in vitro KeratinoSens™ assay conducted according to OECD TG 442D the test material was dissolved in THF. Based on a molecular weight of 324.82 g/mol a stock solution of 200 mM was prepared. Based on the stock solution a set of twelve master solutions in 100 % solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium and a stable suspension was formed. The following concentration range was tested in the assay: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM. Cells were incubated with the test item for 48 h at 37 °C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement. In the first experiment, a max luciferase activity (Imax) induction of 6.41 was determined at a test item concentration of 31.25 µM. The corresponding cell viability was 29.4 %. A significant luciferase induction >1.5 was found in the whole tested concentration range. Therefore, no EC1.5 value could be calculated. A cytotoxic effect was observed from 7.81 µM onwards. In the second experiment, a max luciferase activity (Imax) induction of 3.23 was determined at a test item concentration of 31.25 µM. The corresponding cell viability was 40.6 %. A significant luciferase induction >1.5 was found in the whole tested concentration range. Therefore, no EC1.5value could be calculated. A cytotoxic effect was observed from 7.81 µM onwards. A dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. In this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as sensitiser.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-09-03 to 2018-10-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)

Version / remarks:

25 June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Details of test system:

THP-1 cell line [442E]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: The test item was freshly prepared immediately prior to use. The test item was only soluble tetrahydrofuran (THF) at a concentration of 400 mg/mL. Vortex mixing was used to aid solubilisation. Stock solutions were prepared by diluting the highest soluble concentration seven times with a constant dilution factor of 1:2.
- Preparation of the test chemical serial dilutions: The working stock solutions were prepared by diluting each stock solution 250 times with cell culture medium. Precipitates were observed when diluted 1:250 in cell culture medium. Sonication was used to aid solubilisation. The working stock solutions were applied to the cells by adding equal volumes of each solution to prepared cells, resulting in a further 1:2 dilution of the working solutions. The solvent was present at a constant volume ratio of 0.2 % (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.
- Preparation of the positive controls: 2,4-dinitrochlorobenzene (DNCB) at a final concentration of 4 µg/mL (alternatively at the concentration of the CV75) was tested concurrently with the test item. DNCB was dissolved in DMSO. Starting from 400 mg/mL solution, eight stock solutions (eight concentrations) were prepared, by 2-fold serial dilutions using the corresponding solvent. These stock solutions were further diluted 250-fold into culture medium (working solutions). The working solutions were finally used for treatment by adding an equal volume of working solution to the volume of THP-1 cell suspension in a 96-well plate to achieve a further 2-fold dilution. Dilution resulted in a final DMSO concentration of 0.2 % (v/v).
- Preparation of the solvent, vehicle and negative controls:
Medium Control: A medium control was included in the test.
Solvent Controls: Solvent controls were included in the test. The solvent controls were set up depending on the appropriate solvent. Since the test item was solubilized in THF, a THF control served as solvent control for the test item. Since the positive control was solubilized in DMSO, a DMSO control was included and served as solvent control for the positive control. The solvent controls were diluted according to the same procedure described for the positive control, resulting in a final concentration of 0.2 % (v/v) for THF.
- Stable dispersion obtained: yes
- Log Kow of the test chemical: 7.56

DOSE RANGE FINDING ASSAY:
- Highest concentration used: 800 µg/mL (400 mg/mL stock solution)
- Solubility in solvents: Solubility of the test item was determined prior to the main experiment. The test item was dissolved in 0.9 % NaCl at a final concentration of 500 mg/mL. Test items not soluble in 0.9 % NaCl solution were dissolved in DMSO at a concentration of 500 mg/mL. If the test item was not soluble in DMSO, other solvents (e.g. THF) were used. It was taken care that the test chemical is dissolved or stably dispersed in the chosen solvent and that it does not interfere with the test design. If the test item was not soluble in DMSO or a different organic solvent at 500 mg/mL, the highest soluble concentration was tested by diluting the solution from 500 mg/mL with a constant factor of 1:2 up to a minimal concentration of 1 mg/mL. The test item was only soluble tetrahydrofuran (THF) at a concentration of 400 mg/mL.
- Solubility in incubation medium: not specified
- Results of selecting appropriate concentration and determination of cytotoxicity: Starting from 400 mg/mL solutions of the test chemicals, eight stock solutions (eight concentrations) were prepared, by 2-fold serial dilutions using the corresponding solvent. These stock solutions were further diluted 250-fold into culture medium (working solutions). The working solutions were finally used for treatment by adding an equal volume of working solution to the volume of THP-1 cell suspension in a 96-well plate to achieve a further 2-fold dilution. After cell incubation and staining the PI uptake of the cells and therefore cytotoxicity was analysed immediately by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ > 650 nm. A total of 10,000 living (PI negative) cells were acquired and cell viability was calculated for each test concentration. The CV75 value, i.e. the concentration showing 75 % cell survival, was calculated by log-linear interpolation.
- Final concentration range selected on basis of: The CV75 value was used to calculate the concentration range of the test item for the main experiment. Since there was no cytotoxicity, no CV75 could be derived. The main experiment was performed covering a concentration range from 800 – 223.27 μg/mL (400 – 111.63 mg/mL stock solution).

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 1
- Number of repetitions: 2
- Test chemical concentrations: 800, 666.67, 555.56, 462.96, 385.80, 321.50, 267.92, 223.27 μg/mL
- Application procedure: For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of 0.1 – 0.2 x 10^6 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation (125 x g) and were re-suspended in fresh culture medium at a density of 2 x 10^6 cells/mL. Then, 500 μL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 10^6 cells/well). The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5 % CO2.
- Exposure time: 24 h ± 0.5 h
- Study evaluation and decision criteria used: FACS data analysis was performed using the software BD FACS DIVA 6.0. Further data analysis like calculation of the CV75, calculation of the RFI and calculation of the Effective Concentration 150 and Effective Concentration 200 values were performed using the software Microsoft Excel 2010. The mean values and standard deviations of the single replicates were determined using the respective excel commands. For criteria please refer to “Prediction model” below
- Description on study acceptance criteria: The test meets acceptance criteria if: - the cell viability of the solvent controls is >90 %, - the cell viability of at least four tested doses of the test item in each run is >50 %, - the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200 % for CD54 at a cell viability of >50 %, - the RFI values of the solvent control is not ≥150 % for CD86 and not ≥200 % for CD54, - the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105 %.

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density): passage 25 (first experiment) and 26 (second experiment), cell suspension were seeded into a 24 well flat-bottom plate (1 x 10^6 cells/well)
- Incubation conditions: 37 °C ± 1 °C and 5 % CO2
- Washing conditions: Cells were washed twice with FACS buffer. It was carried out on ice with pre-cooled buffer.

MEASUREMENT OF CELL SURFACE EXPRESSION/LUCIFERASE ACTIVITY
For h-CLAT and USENS
- Flow cytometry used: FACS: BD Canto II, Software: BD FACS DIVA 6.0, Voltage Settings: FSC: 300 V SSC: 250 V FITC: 400 V PI: 420 V, Threshold value of FSC: 5000, Compensation: PI - 14 % FITC FITC – 0 % PI
- Plate used: 96-well V-bottom plate
- Propidium iodide staining/cytotoxicity measurements: PI staining was done just prior to the measurement by adding PI solutions to each sample (final concentration of PI was 0.625 μg/mL).
- Preparation for CD54 and/or CD86 expression measurements/cell staining: After washing, cells were blocked using 600 μL of a FcR blocking buffer (FACS buffer containing 0.01 % (w/v) Globulin Cohn Fraction) and incubated at 4 °C for 15 min. After blocking, cells were split in three aliquots into a 96-well V-bottom plate. After centrifugation (approx. 250 x g), cells were stained with 50 μL of FITC-labelled anti-CD86, anti-CD54, or mouse IgG1 (isotype) antibodies in the dark for 30 min. All antibodies were diluted in FACS buffer at an appropriate manner. The expression levels of CD86 and CD54 as well as cell viability were analysed by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ = 530 nm ± 15 nm for FITC and λ > 650 nm for PI. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 were calculated.

DATA EVALUATION
- Cytotoxicity assessment: Done by measuring cell viability
- Prediction model used: For CD86/CD54 expression measurement, each test item was tested in at least two independent runs to derive a single prediction. Each independent run was performed on a different day or on the same day provided that for each run: independent fresh stock solutions and working solutions of the test chemicals and antibody solutions were prepared and independently harvested cells were used. Sensitising potential of the test item was predicted from the mean percentage expression of CD86 and CD54. Any test chemical tested by the h-CLAT was considered positive if the RFI of CD86 was equal to or greater than 150 % at any tested dose at a cell viability ≥ 50 % in at least two independent runs or if the RFI of CD54 was equal to or greater than 200 % at any tested dose at a cell viability ≥ 50 % in at least two independent runs or if the RFIs of both the CD86 and CD54 were equal to or are greater than 150 % and 200 % respectively at any tested dose at a cell viability ≥ 50 % in at least two independent runs. In case of not concordant results a third run should be conducted to make the final prediction. Otherwise the results were considered as inconclusive. A negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x CV75 is <90 %. In contrast, a positive test outcome was accepted irrespective of cell viabilities >90 % at a concentration of 1.2 x CV75. If no CV75 could be derived negative test results can be accepted when the test item is tested at the highest soluble concentration (5000 µg/mL for 0.9 % NaCl solution; 1000 µg/mL for DMSO or a different organic solvent) even if the cell viability is >90 %.

Vehicle / solvent control:

other: 0.2 % DMSO (v/v) in cell culture medium 0.2 % THF (v/v) in cell culture medium

Negative control:

not applicable

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Positive control results:

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150 % for CD86 (RFI 310 % experiment 1; 314 % experiment 2) and 200 % for CD54 (232 % experiment 1; 256 % experiment 2) were clearly exceeded.

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

other: RFI > 150 (CD86)

Value:

157 %

Cell viability:

93.4

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

other: max. RFI for CD54

Value:

78 %

Cell viability:

95.6 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: observed at concentration of 223.27 µg/mL

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

other: RFI > 150 (CD86)

Value:

152 %

Cell viability:

91.3 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

other: max. RFI for CD54

Value:

73 %

Cell viability:

91.4 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: observed at concentration of 223.27 µg/mL

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

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

DEMONSTRATION OF TECHNICAL PROFICIENCY: not specified

ACCEPTANCE OF RESULTS:
Please refer to "Any other information on results".

Results of the Cell Batch Activation Test

Sample	Concentration [µg/mL]	CD86			CD54			Activated	Pass /Fail
		Cell Viability [%]	RFI	Threshold OECD TG 442E	Cell Viability [%]	RFI	Threshold OECD TG 442E	yes/no
DNCB	4 µg/mL	87.5	373	>150	88.1	358	>200	yes	pass
NiSO4	100 µg/mL	83.5	295	>150	82.1	603	>200	yes	pass
LA	1000 µg/mL	95.7	81	≤150	95.8	100	≤200	no	pass

Results of the Dose Finding Assay

Sample		Experiment 1
		Concentration applied [µg/mL]	Cell Viability [%]
Medium Control	--	--	95.00
Solvent Control	THF	--	95.60
Test material	C8	6.25	93.80
	C7	12.50	94.80
	C6	25.00	94.40
	C5	50.00	94.10
	C4	100.00	93.30
	C3	200.00	92.30
	C2	400.00	94.10
	C1	800.00	94.30
Calculated CV75 [µg/mL]		No CV75
Mean CV75 [µg/mL]		No CV75
SD CV 75 [µg/mL]		No SD

CD54 and CD86 Expression Experiment 1

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	97.2	97.2	96.6	3303	1165	606	2697	559	91	73	545	192
Solvent Control 2 (THF)	0.20%	97.0	96.6	96.7	3221.0	1150.0	624.0	2597	526	100	100	516	184
Solvent Control 1 (DMSO)	0.20%	95.9	96.9	96.5	3525	1332	569	2956	763	100	100	620	234
DNCB	4.00	86.7	87.0	87.3	9787	2391	624	9163	1767	310	232	1568	383
Test material	800	93.7	93.3	93.2	4620	890	552	4068	338	157	64	837	161
	666.67	93.5	93.2	93.3	4635	921	561	4074	360	157	68	826	164
	555.56	94.9	94.4	95.2	3888	955	550	3338	405	129	77	707	174
	462.96	95.0	95.2	95.4	3963	963	557	3406	406	131	77	711	173
	385.80	95.3	95.1	91.3	3792	957	624	3168	333	122	63	608	153
	321.50	93.2	93.5	93.0	4087	916	552	3535	364	136	69	740	166
	267.92	95.3	95.2	95.8	3642	946	563	3079	383	119	73	647	168
	223.27	95.7	95.6	95.5	3671	971	561	3110	410	120	78	654	173

CD54 and CD86 Expression Experiment 2

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	C86	CD54
Medium Control	-	94.4	94.7	93.8	4269	1460	585	3684	875	94	117	730	250
Solvent Control 2 (THF)	0.20%	95.1	94.8	95.2	3974	1398	605	3369	793	100	100	657	231
Solvent Control 1 (DMSO)	0.20%	94.1	94.2	93.9	4492	1336	585	3907	751	100	100	768	228
DNCB	4.0	80.4	79.5	79.2	12892	2541	622	12270	1919	314	256	2073	409
Test material	800	88.10	86.70	87.00	6865	1089	617	6248	472	185	60	1113	176
	666.67	88.50	88.70	89.10	6594	1071	591	6003	480	178	61	1116	181
	555.56	88.40	89.10	89.30	6589	1164	618	5971	546	177	69	1066	188
	462.96	91.10	91.00	90.70	6055	1114	593	5462	521	162	66	1021	188
	385.80	90.00	90.60	89.50	5808	1108	600	5208	508	155	64	968	185
	321.50	91.20	91.00	91.80	5689	1023	583	5106	440	152	55	976	175
	267.92	91.40	91.00	91.60	5494	1036	581	4913	455	146	57	946	178
	223.27	91.70	91.20	91.30	5515	1189	613	4902	576	146	73	900	194

Acceptance Criteria

Acceptance Criterion	Range	Experiment 1			pass/fail	Experiment 2			pass/fail
cell viability solvent controls [%]	>90	95.9	-	97.2	pass	93.8	-	95.2	pass
number of test dosed with viability >50% CD86	≥4	8			pass	8			pass
number of test dosed with viability >50% CD54	≥4	8			pass	8			pass
number of test dosed with viability >50% IgG1	≥4	8			pass	8			pass
RFI of positive control of CD86	≥150	310			pass	314			pass
RFI of positive control of CD54	≥200	232			pass	256			pass
RFI of DMSO control of CD86	<150	110			pass	106			pass
RFI of DMSO control of CD54	<200	136			pass	86			pass
RFI of THF solvent control of CD86	<150	96			pass	91			pass
RFI of THF solvent control of CD54	<200	94			pass	91			pass
MFI ratio CD86/IgG1 for medium control [%]	>105	545			pass	730			pass
MFI ratio CD86/IgG1 for DMSO control [%]	>105	620			pass	768			pass
MFI ratio CD86/IgG1 for THF control [%]	>105	516			pass	657			pass
MFI ratio CD54/IgG1for medium control [%]	>105	192			pass	250			pass
MFI ratio CD54/IgG1for DMSO control [%]	>105	234			pass	228			pass
MFI ratio CD54/IgG1for THF control [%]	>105	184			pass	231			pass

Historical Data

Criterion	mean	SD	N
cell viability solvent controls [%]	96.2	1.6	216
number of test doses with viability >50%	-	-	555
RFI of positive control of CD86	358.9	90.1	36
RFI of positive control of CD54	435.7	285.8	36
RFI of solvent control (THF) of CD86	102.9	17.4	36
RFI of solvent control (THF) of CD54	102.0	15.4	36
MFICD86 / MFIIgG1for medium control [%]	285.6	124.3	36
MFICD86 / MFIIgG1for THF control [%]	270.8	105.0	36
MFICD54 / MFIIgG1for medium control [%]	308.6	137.1	36
MFICD54 / MFIIgG1for THF control [%]	158.2	28.4	36

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In this study under the given conditions the test item did upregulate the cell surface marker CD86 in at two independent experiment runs. Therefore, the test item is considered to be a skin sensitiser.

Executive summary:

A study according to OECD TG 442E (h-CLAT) was conducted to evalute the potential of the test item to active dentritic cells. In the study the test material was dissolved in THF. For the dose finding assay stock solutions starting with the highest soluble concentrationfrom 400 mg/mL to 3.13 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37 °C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis. Due to a lack of cytotoxicity, no CV75 could be derived. Therefore, the main experiment was performed covering the following concentration steps: 800, 666.67, 555.56, 462.96, 385.80, 321.50, 267.92, 223.27 µg/mL In all experiments precipitation of the test item was observed for all concentration steps when mixing the test item stock solutions with cell culture medium. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining. No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was 93.7 % (CD86), 93.3 % (CD54) and 93.2 % (isotype IgG1 control) in the first experiment and 88.1 % (CD86), 86.7 % (CD54) and 87.0 % (isotype IgG1 control) in the second experiment. The controls confirmed the validity of the study for all experiments. The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150 % for CD86 (RFI 310 % experiment 1; 314 % experiment 2) and 200 % for CD54 (232 % experiment 1; 256 % experiment 2) were clearly exceeded. The expression of the cell surface marker CD86 was upregulated to 157 % (800 µg/mL and 666.67 µg/mL) in the first experiment and in almost all concentrations in the second experiment, except for the two lowest concentrations 267.92 µg/mL and 223.27 µg/mL (146 %). In contrast, the expression of the cell surface marker CD54 was not upregulated above the threshold of 200 % in any of the experiments. Since one of the cell surface markers clearly exceeded the threshold in two independent experiments the test item is considered to be a skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

To evaluate the potential of the test item to cause skin sensitisation an in vitro/ in chemico testing battery based on the OECD Adverse Outcome Pathway (AOP) for the assessment of the skin sensitisation potential of chemicals was performed. The predictions of the conducted tests are used together in a weight of evidence approach to derive a conclusion on the potential of the test item to cause skin sensitisation.

 

OECD TG 442C (reference 7.4.1-1)

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine. In the present study, according to OECD TG 442C, the test item was dissolved in N.N-dimethylformamide (DMF) based on the results of the pre-experiments. Based on a molecular weight of 324.82 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC. For the 100 mM solution of the test item precipitation was observed when diluted with the cysteine peptide solution. After the 24 h± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged prior to the HPLC analysis. For the 100 mM stock solution of the test item precipitation was observed when diluted with the lysine peptide solution. After the 24 h± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged prior to the HPLC analysis. Phase separation (droplets) was observed for the co-elution control of the positive control. The sample was not centrifuged prior to the HPLC analysis. A minor co-elution of the test item with the lysine peptide peak was observed. The peak area determined in the co-elution controls corresponds to 0.0104 mM of lysine peptide. Therefore, the given peak areas and corresponding lysine peptide values can only be considered as an estimation of the peptide depletion and cannot be used for evaluation. The 100 mM stock solution of the test item showed minimal reactivity towards the cysteine peptide. The mean depletion of the cysteine peptide was ≤ 13.89 % (5.58 %). According to the evaluation criteria in the guideline, if a precipitation or phase separation is observed after the incubation period, peptide depletion may be underestimated and a conclusion on the lack of reactivity cannot be drawn with sufficient confidence in case of a negative result. Due to the observed precipitation in the cysteine experiment no prediction can be made. Since precipitation was observed for the lysine and cysteine peptide samples containing also test item after the incubation period, no final prediction can be made. In this study under the given conditions the test item showed minimal reactivity towards the cysteine peptide. Due to the observed precipitation in the cysteine peptide experiment a prediction cannot be made.

 

OECD TG 442D (reference 7.4.1-2)

In the in vitro KeratinoSens™ assay conducted according to OECD TG 442D the test material was dissolved in THF. Based on a molecular weight of 324.82 g/mol a stock solution of 200 mM was prepared. Based on the stock solution a set of twelve master solutions in 100 % solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium and a stable suspension was formed. The following concentration range was tested in the assay: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM. Cells were incubated with the test item for 48 h at 37 °C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement. In the first experiment, a max luciferase activity (Imax) induction of 6.41 was determined at a test item concentration of 31.25 µM. The corresponding cell viability was 29.4 %. A significant luciferase induction >1.5 was found in the whole tested concentration range. Therefore, no EC1.5 value could be calculated. A cytotoxic effect was observed from 7.81 µM onwards. In the second experiment, a max luciferase activity (Imax) induction of 3.23 was determined at a test item concentration of 31.25 µM. The corresponding cell viability was 40.6 %. A significant luciferase induction >1.5 was found in the whole tested concentration range. Therefore, no EC1.5value could be calculated. A cytotoxic effect was observed from 7.81 µM onwards. A dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. In this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as sensitiser.

 

OECD TG 442E (reference 7.4.1-3)

A study according to OECD TG 442E (h-CLAT) was conducted to evalute the potential of the test item to active dentritic cells. In the study the test material was dissolved in THF. For the dose finding assay stock solutions starting with the highest soluble concentrationfrom 400 mg/mL to 3.13 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37 °C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis. Due to a lack of cytotoxicity, no CV75 could be derived. Therefore, the main experiment was performed covering the following concentration steps: 800, 666.67, 555.56, 462.96, 385.80, 321.50, 267.92, 223.27 µg/mL In all experiments precipitation of the test item was observed for all concentration steps when mixing the test item stock solutions with cell culture medium. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining. No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was 93.7 % (CD86), 93.3 % (CD54) and 93.2 % (isotype IgG1 control) in the first experiment and 88.1 % (CD86), 86.7 % (CD54) and 87.0 % (isotype IgG1 control) in the second experiment. The controls confirmed the validity of the study for all experiments. The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150 % for CD86 (RFI 310 % experiment 1; 314 % experiment 2) and 200 % for CD54 (232 % experiment 1; 256 % experiment 2) were clearly exceeded. The expression of the cell surface marker CD86 was upregulated to 157 % (800 µg/mL and 666.67 µg/mL) in the first experiment and in almost all concentrations in the second experiment, except for the two lowest concentrations 267.92 µg/mL and 223.27 µg/mL (146 %). In contrast, the expression of the cell surface marker CD54 was not upregulated above the threshold of 200 % in any of the experiments. Since one of the cell surface markers clearly exceeded the threshold in two independent experiments the test item is considered to be a skin sensitiser.

 

Conclusion

A DPRA was conducted to address the molecular initiating event of the AOP, namely protein reactivity, by quantifying the reactivity of test chemicals towards synthetic model peptides containing either lysine or cysteine. The assay showed minimal reactivity towards the cysteine peptide. However, as precipitation was observed for the lysine and cysteine peptide samples containing test item, no final prediction could be derived for the first key event of the AOP.

The in vitro KeratinoSens™ assay was used to address the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs, thus, leading to a positive prediction for the second key event of the AOP.

An in vitro human cell line activation test (h-CLAT) was conducted to evaluate the potential of the test item to induce dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface marker CD86 was upregulated by the test item. Therefore, the test item was considered positive for the third molecular key event of the AOP.

The predictions based on the key events of the AOP show that the test item possess a skin sensitisation potential. Therefore, the test is determined to be a skin sensitizer using the weight of evidence approach.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.  Based on available data on skin sensitisation, the test item requires classification for causing skin sensitisation (Category 1, H317: May cause an allergic skin reaction) according to Regulation (EC) No 1272/2008 (CLP).