[No public or meaningful name is available]

Endpoint summary

Description of key information

three in vitro/in chemico tests available on skin sensitisation

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

09 - 18 May 2018

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:

adopted Feb 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU-Method B.60 of the Commission Regulation (EU) No. 2017/735: “In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method”

Version / remarks:

adopted Feb 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt Rheinland-Pfalz, Mainz, Germany

Type of study:

activation of keratinocytes

Details on the study design:

TEST CELL LINE
- Type: LuSens cell line
- Passage number: 8 (cytotoxicity range finder assay), 10 (experiment I and II)

CELL CULTURE CONDITIONS
- Type and identity of media:
Maintenance medium: Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 9% fetal bovine calf serum
- Temperature (°C): 37 ± 1.0
- CO2 (%): 5.0 ± 0.5

TEST CONCENTRATIONS
0.2, 0.4, 0.8, 1.6, 3.1, 6.3, 12.5, 25, 50, 100, 200 and 400 μg/mL (cytotoxicity range finder assay)
53.8, 64.6, 77.5, 93.0, 111.6, 134.0, 160.8, 192.9, 231.5, 277.8, 333.3 and 400.0 μg/mL (experiment I and II)

CONTROLS
Solvent control:
- Substance: DMEM
Positive control:
- Substance: p-phenylenediamine
- Final concentration: 80 µM
Negative control:
- Substance: DL-lactic acid
- Final concentration: 5000 µM

EXPOSURE CONDITIONS
- Exposure duration: 48 h (cytotoxicity range finder assay); 24 h (experiment I) and 25 and 45 min (experiment II)
- Temperature (°C): 37 ± 1.0
- CO2 (%): 5.0 ± 0.5

NUMBER OF REPLICATIONS: triplicates in two independent experiments

DETERMINATION OF CELL VIABILITY
- Method: MTT assay
- MTT concentration: 5 mg/mL
- Incubation time: 2 h
- Device: plate reader
- Wavelength: 600 nm
- Temperature (°C): 37 ± 1.0
- CO2 (%): 5.0 ± 0.5

ACCEPTANCE CRITERIA
- The average induction for the positive control should be ≥ 2.5 fold and it should have a relative viability of at least 70%.
- The induction triggered by the negative control and growth control should be < 1.5 fold as compared to the induction of the solvent control and the viability should be above 70%.
- The average percentage standard deviation (luciferase induction) of the variability in at least 21 solvent control wells should be below 20%.
- At least 3 test concentrations must be within viability limits, i.e. have relative viability of at least 70%.

EVALUATION CRITERIA
A test compound is considered to have the potential to activate the Nrf2 transcription factor if the luciferase induction is ≥ 1.5 fold and statistically significant compared to the vehicle control in 2 (or more than) consecutive non-cytotoxic (relative viability ≥ 70%) tested concentrations whereby at least three tested concentrations must be non-cytotoxic in two independent valid experiments.
A test compound is considered not to have the potential to activate the Nrf2 transcription factor if the effects mentioned above are not observed. A negative result obtained with test chemicals that do not form a stable dispersion and/or were not tested up to 2000 μM (or 2000 μg/mL for test chemicals with no defined molecular weight) and for which no cytotoxicity is observed in any of the tested concentration should be considered as inconclusive.
In order to come to a conclusion on the skin sensitization hazard of a substance, a minimum of two valid and independent experiments needs to indicate a positive or negative result according to the above-described criteria. If the first two experiments come to the same result (i.e. either being negative or being positive) no further testing is required. In case that the first two experiments give discordant results (i.e. one is negative and the other is positive), a third independent experiment needs to be conducted to complete the study. The skin sensitizing potential (corresponding to the potential to activate the Nrf2 transcription factor) of a test substance is determined by the result of the majority of the repetitions of an experiment. If two of two or two of three experiments are negative/positive, the substance is considered as negative/positive.

Positive control results:

The positive control p-phenylenediamine induced a clear effect with an induction value of 5.9 fold in comparison to the solvent control for both experiments. The relative viability was 85.4 and 84.9% in experiment I and II, respectively.

Key result

Run / experiment:

other: Experiment I and II

Parameter:

other: luciferase induction

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: induction is reported as "x fold induction" therefore no unit is provided

Other effects / acceptance of results:

No significant reduction of growth was observed in all tested test item concentrations. Therefore, all tested concentrations could be evaluated for luciferase induction. In all tested concentrations of the test item no substantial and reproducible dose dependent increase of luciferase induction was measured.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative and growth control induced a 1.0- and 1.1-fold induction in experiment I and a 1.0- and 1.0-fold induction in experiment II, respectively. The cell viabilities were 103.6, 99.9, 105.1 and 101.2%. Since the average induction of the negative and growth control were < 1.5 fold and the viability was at least 70%, the egative and growth control results were considered to be valid.
- Acceptance criteria met for positive control: The positive control p-phenylenediamine induced a clear effect with an induction value of 5.9 fold in comparison to the solvent control for both experiments. The relative viability was 85.4 and 84.9% in experiment I and II, respectively. Since the average induction of the positive control was ≥ 2.5 fold and the viability was at least 70%, the positive control results were considered to be valid.
- Acceptance criteria met for variability between replicate measurements: The average percentage standard deviation was 6.02 and 6.88% in experiment I and II, respectively, which was below 20%. Since variation between the replicates was less than 20%, results of this run were considered as valid.

Table 1: Results of Experiment I

		Induction of Luciferase			Viability of the Cells
Parameter	Concentration	Induction	Standard Deviation	Standard Deviation	Relative Viability	Standard Deviation	Standard Deviation
	[µg/mL]	fold		[%]	[%]		[%]
Solvent Control	-	1.0	0.06	6.02	100.0	2.22	2.22
Growth Control	-	1.0	0.07	7.14	99.9	3.91	3.91
Negative Control	5000 µM	1.1	0.05	5.01	103.6	2.24	2.16
Positive Control	80 µM	5.9	0.28	4.74	85.4	2.26	2.65
Test item	53.8	1.0	0.14	13.57	104.5	1.94	1.86
Test item	64.6	1.1	0.09	8.65	99.2	0.45	0.46
Test item	77.5	1.0	0.04	3.69	98.3	1.20	1.22
Test item	93.0	1.1	0.05	4.58	99.0	2.25	2.28
Test item	111.6	1.0	0.02	1.99	97.8	2.85	2.92
Test item	134.0	1.1	0.12	10.90	96.9	2.94	3.03
Test item	160.8	1.1	0.06	6.09	97.8	2.96	3.03
Test item	192.9	1.0	0.07	6.44	96.5	3.75	3.88
Test item	231.5	1.1	0.06	5.38	96.0	4.04	4.20
Test item	277.8	1.1	0.07	6.20	97.6	1.54	1.58
Test item	333.3	1.0	0.03	2.87	98.1	1.70	1.73
Test item	400.0	1.1	0.01	0.86	99.0	1.14	1.15

Table 2: Results of Experiment II

		Induction of Luciferase			Viability of the Cells
Parameter	Concentration	Induction	Standard Deviation	Standard Deviation	Relative Viability	Standard Deviation	Standard Deviation
	[µg/mL]	fold		[%]	[%]		[%]
Solvent Control	-	1.0	0.07	6.88	100.0	2.39	2.39
Growth Control	-	1.0	0.06	5.54	101.2	3.04	3.01
Negative Control	5000 µM	1.0	0.06	5.95	105.1	2.80	2.66
Positive Control	80 µM	5.9	0.59	9.94	84.9	3.01	3.55
Test item	53.8	1.1	0.08	7.66	105.4	0.56	0.53
Test item	64.6	1.1	0.04	4.08	99.4	1.47	1.47
Test item	77.5	1.1	0.02	2.16	99.1	0.90	0.91
Test item	93.0	1.0	0.05	4.54	97.9	1.32	1.34
Test item	111.6	1.0	0.04	3.68	95.9	4.17	4.35
Test item	134.0	1.1	0.06	5.70	96.4	6.49	6.74
Test item	160.8	1.1	0.03	2.64	97.3	2.44	2.51
Test item	192.9	1.0	0.04	4.39	98.7	1.91	1.94
Test item	231.5	1.1	0.04	4.00	99.3	2.77	2.79
Test item	277.8	0.9	0.04	4.58	97.8	1.19	1.21
Test item	333.3	1.1	0.01	0.84	97.7	2.25	2.30
Test item	400.0	1.1	0.04	4.07	99.4	2.37	2.38

Interpretation of results:

other: no skin sensitising potential based on the key event "activation of keratinocytes"

Conclusions:

Under the conditions of the test, it can be concluded, that the test substance is no sensitiser in KeratinoSens Assay. The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.

Since 2 out of 3 key events resulted in a positive result, the test substance was considered to be a skin sensitiser. The available data meet the criteria for classification in Skin Sens. 1, H317 according to Regulation (EC) No. 1272/2008.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

14 Feb - 14 Apr 2018

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:

adopted Feb 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Rheinland-Pfalz, Germany

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

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.

TEST METHOD
The DPRA is an in chemico method, which quantifies the remaining concentration of cysteine or lysine containing peptide, following 22 hours incubation with the test item at 25 ± 2.5 °C. Relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 and 258 nm. Cysteine and lysine peptide percent depletion values are calculated and used in a prediction model which allow assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

TEST SYSTEM
- Supplier of synthetic peptides: Genecust, Dudelange, Luxemburg
- Peptide stock solution preparation: Stock solutions of each peptide were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in appropriate buffer solution.
Cysteine-containing peptide: 0.669 mM Cys-Peptide solution was prepared by dissolving 22.6 mg of the peptide in 45.0 mL phosphate buffer, pH 7.5 (batch no. 20180215 and batch no. 20180308).
Lysine-containing peptide: 0.667 mM Lys-Peptide solution was prepared by dissolving 23.3 mg of the peptide in 45.0 mL ammonium acetate buffer, pH 10.2 (batch no. 20180214 and batch no. 20180312) and by dissolving 15.5 mg of the peptide in 30.0 mL ammonium acetate buffer, pH 10.2. (batch no. 20180412).

POSITIVE CONTROL
- Cinnamaldehyde (CAS 104-55-2, 99.1%, batch no. MKBT8955V) was used as 100 mM solution in acetonitrile for the Cys-peptide.
- 2,3-Butanedione (CAS 431-03-8, 99.4%, batch no. BCBS3560V) was used as 100 mM solution in acetonitrile for the Lys-peptide.
As cinnamaldehyde mixed with the lysine peptide turned turbid in all experiments performed during the implementation phase, it was considered unsuitable as positive control. Instead, the proficiency chemical 2,3-butanedione is used as positive control showing mid-range depletion for the lysine peptide.

CO-ELUTION CONTROL
- Co-elution control was prepared without peptide, to verify if the test item absorbs at 220 and 258 nm and has a similar retention time as a peptide and/or interfere with the data analysis.

REFERENCE CONTROLS
For both peptides, four sets of solvent controls using acetonitrile instead of test item stock solution were prepared in triplicate (sets A, B1, B2 and C, total 12 samples per peptide). Set A was analysed together with the peptide calibration standards, sets B1 and B2 were analysed at the start and end of the analysis sequence and were used as stability control for the peptide over the total analysis time. Set C was incubated and analysed together with the samples and was used for calculation of the peptide depletion of positive controls.

TEST SUBSTANCE PREPARATION
The test substance was prepared as a 100 mM solution in acetonitrile.

INCUBATION CONDITIONS
- Peptide ratios: cysteine-containing peptide: 1:10; lysine-containing peptide: 1:50
- Temperature used during treatment / exposure: 25 ± 2.5 °C
- Duration of treatment / exposure: minimum of 22 h

NUMBER OF REPLICATES
for each peptide triplicates were prepared for test substance and controls

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Agilent Technologies with UV Detector
- Analytical Column: ACE Excel SuperC18 150x3 mm column with 3 µm particles and pre-column Phenomenex Security Guard C18, 4x3 mm
- HPLC mobile phase:
A: 0.1% (v/v) trifluoracetic acid in water (HPLC grade)
B: 0.085% (v/v) trifluoracetic acid in acetonitrile (HPLC grade)
- Flow: 0.55 mL/min
- Column temperature: 30 ± 2 °C
- Gradient:
Time (min): 0, 10, 10.5, 12, 13, 20
% B: 10, 25, 90, 90, 10, 10
- Wavelength: 220 and 258 nm
- Injection volume: 7 μL
- peptide standards: calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, and a buffer blank were measured.

Positive control results:

Mean peptide depletion (%) of the positive control was 31.57, 26.01 and 34.53% in all three experiments for the lysine peptide, respectively, and 77.61 and 81.40% in both experiments for the cysteine peptide, respectively.

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: mean peptide depletion

Value:

7.81 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: Mean peptide depletion

Value:

7.83 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS
- The mean cysteine peptide depletion and SD of the three replicates of the positive control cinnamaldehyde were 77.61% ± 2.46% and 81.40% ± 2.00% for both experiments, and thus in the acceptable range as defined in the acceptability criteria.
- The mean lysine peptide depletion and SD of the three replicates of the positive control 2,3-butanedione were 31.57% ± 1.48%, 26.01% ± 2.69% and 34.53% ± 2.31% for the three experiments, and thus in the acceptable range as defined in the acceptability criteria.
- The SD for the test item replicates were 1.42 and 3.75%, respectively, and thus < 14.9% in both experiments with the cysteine peptide.
- The SD for the test item replicates were 5.38, 5.06 and 2.40%, respectively, and thus < 11.6% in all three experiments with the lysine peptide.

The mean peptide depletion in the Lys-peptide and Cys-peptide assay was 7.81% in the first experiment. The value was verified with a mean depletion of 7.83% in the second experiment, therefore the test item was classified with:
DPRA Prediction: positive
Reactivity class: low

Table 1: Calculated peptide depletion values for the Lys-Peptide (Experiment 1)

Sample name	Depletion [%]
	Single	Mean	SD
Positive control Rep. 1	30.20	31.57	1.48
Positive control Rep. 2	31.39
Positive control Rep. 3	33.14
Test item Rep. 1	4.04	4.90	5.38
Test item Rep. 2	0.00
Test item Rep. 3	10.65

Rep.: Replicate

SD: Standard deviation

Table 2: Calculated peptide depletion values for the Lys-Peptide (Experiment 2)

Sample name	Depletion [%]
	Single	Mean	SD
Positive control Rep. 1	24.28	26.01	2.69
Positive control Rep. 2	24.64
Positive control Rep. 3	29.11
Test item Rep. 1	1.57	3.68	5.06
Test item Rep. 2	0.00
Test item Rep. 3	9.45

Rep.: Replicate

SD: Standard deviation

Table 3: Calculated peptide depletion values for the Lys-Peptide (Experiment 3)

Sample name	Depletion [%]
	Single	Mean	SD
Positive control Rep. 1	32.96	34.53	2.17
Positive control Rep. 2	33.64
Positive control Rep. 3	37.00
Test item Rep. 1	3.00	4.79	2.40
Test item Rep. 2	7.51
Test item Rep. 3	3.85

Rep.: Replicate

SD: Standard deviation

Table 4: Calculated peptide depletion values for the Cys-Peptide (Experiment 1)

Sample name	Depletion [%]
	Single	Mean	SD
Positive control Rep. 1	74.78	77.61	2.46
Positive control Rep. 2	78.77
Positive control Rep. 3	79.27
Test item Rep. 1	9.85	10.71	1.42
Test item Rep. 2	9.94
Test item Rep. 3	12.35

Rep.: Replicate

SD: Standard deviation

Table 5: Calculated peptide depletion values for the Cys-Peptide (Experiment 2)

Sample name	Depletion [%]
	Single	Mean*	SD
Positive control Rep. 1	79.37	81.40	2.00
Positive control Rep. 2	81.47
Positive control Rep. 3	83.37
Test item Rep. 1	8.24	11.98	3.75
Test item Rep. 2	11.98
Test item Rep. 3	15.74

Rep.: Replicate

SD: Standard deviation

Interpretation of results:

other: "low" skin sensitising potential based on the key event "protein reactivity"

Conclusions:

Under the conditions of the test, the test substance showed reactivity towards selected proteins. The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.

Since 2 out of 3 key events resulted in a positive result, the test substance was considered to be a skin sensitiser. The available data meet the criteria for classification in Skin Sens. 1, H317 according to Regulation (EC) No. 1272/2008.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

06 - 31 Aug 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))

Version / remarks:

adopted Jun 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt Rheinland-Pfalz, Mainz, Germany

Type of study:

activation of dendritic cells

Details on the study design:

TEST CELL LINE
- Source: THP-1 cells, stored in the cell bank of the testing facility and purchased from CLS (Eppenheim, Germany)
- Passage number: 14, 15, 17 and 25

CELL CULTURE CONDITIONS
- Type and identity of media: RPMI-1640 supplemented with 10% fetal calf serum, 2 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol and 100 U/mL penicillin/100 µg/mL streptomycin
- Temperature (°C): 37 ± 1
- CO2 (%): 5.0 ± 0.5

CONTROLS
Negative control
- Substance: culture medium
Solvent control:
- Substance: dimethyl sulfoxide (solvent of the positive control) and RPMI 1640 (solvent of the test item)
Positive control
- Substance: 0.2% 2,4-dinitrochlorobenzene (DNCB)

EXPOSURE CONDITIONS
- Exposure duration: 24 ± 0.5 h

TEST CONCENTRATIONS: In the dose range finding assay the following concentrations 7.8, 15.6, 31.3, 62.5, 125, 250, 500 and 1000 μg/mL were tested to determine the cytotoxic potential of the test substance. Based on the results of the dose range finding assay, the following concentrations 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL were used for the measurement of the expression levels of CD86/CD54.

NUMBER OF REPLICATIONS: single measurement in two independent experiments

CYTOTOXICITY
- Method: Cytotoxicity was determined using propidium iodide staining by flow cytometry.

MEASUREMENT
- Device: BD FACSLyric™ by Becton Dickenson GmbH

STAINING
- Antibodies: FITC (Fluorescein isothiocyanate)-anti-human-CD86, Clone: Fun-1, supplier BD-PharMingen; FITC-anti-human-CD54, Clone: 6.5B5, supplier DAKO; FITC mouse IgG1 (= Isotype control) supplier DAKO; Globulin, Cohn fraction II, III, Human, supplier Sigma

ACCEPTANCE CRITERIA
i. The cell viabilities of medium and solvent/vehicle controls should be higher than 90%.
ii. In the solvent/vehicle control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥ 200%).
iii. For both medium and solvent/vehicle controls, the MFI ratio of both CD86 and CD54 to isotype control should be > 105%.
iv. In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%) and cell viability should be more than 50%.
v. For the test item, the cell viability should be more than 50% in at least four tested concentrations in each run.
If any of these criteria is not met, the experiment is considered not valid and needs to be repeated.

EVALUATION CRITERIA
For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction. An h-CLAT prediction is considered positive if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs, otherwise the h-CLAT prediction is considered negative, if:
− The RFI of CD86 is ≥ than 150 % at any tested concentration with cell viability ≥ 50 %
− The RFI of CD54 is ≥ than 200 % at any tested concentration with cell viability ≥ 50 %
If the first two runs are concordant for both markers, a third experiment is not necessary. Then, the study is completed. If the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 and the other is only positive for CD54, a third run is required. If this third run is negative for both markers, the h-CLAT prediction is considered negative. On the other hand, if the third run is positive for either marker or for both markers, the h-CLAT prediction is considered positive.
In case of a negative result, special care should be taken if the test item
− has a Log Kow > 3.5. Those results should not be considered. However, positive results obtained with those test chemicals can be used for ana
− is a pro-hapten or a pre-hapten
− has an autofluorescence and is emitting at the same wavelength as FITC or as PI

Positive control results:

The RFI values of positive control 2,4-Dinitrochlorobenzene in both experiments were found to be 395 and 7108% for CD86 marker and 206 and 439% for CD54 marker, which met the assay acceptance criteria (RFI CD86 ≥ 150%, RFI CD54 ≥ 200%). The cell viability of positive control was >80% in both the experiments.

Key result

Run / experiment:

other: Experiment I: 3472.2, 4166.7 and 5000 µg/mL

Parameter:

other: RFI CD54

Value:

204 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment I: 3472.2 and 4166.7 µg/mL

Parameter:

other: RFI CD86

Value:

206 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment IV: 3427.2, 4166.7 and 5000 µg/mL

Parameter:

other: RFI CD54

Value:

228 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment IV: 3472.2, 4166.7 and 5000 µg/mL

Parameter:

other: RFI CD86

Value:

257 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER:
Pre-test:
None of the controls induced a cytotoxic effect in the first pre-test. Since the cell viabilities of medium and solvents controls were higher than 90% and the viability of all test item concentrations were >50%, the first pre-test was valid. Since none of the tested concentrations induced a cytotoxic effect with RPMI 1640 as solvent and the test item was soluble at the concentration 500 mg/mL a second pre-test (pre-test 2) using the following concentrations was performed:
5000, 2500, 1250, 625, 312.5, 156.3, 78.1 and 39.1 μg/mL. In the pre-test 2 again none of the controls induced a cytotoxic effect, the cell viabilities of medium and solvent controls were higher than 90% and the viability of all test item concentrations were > 50%, the pre-test 2 was valid.
Main test:
Experiment II did not fulfil the acceptance criteria. The RFI value of CD54 of the positive control was 199 (lower than 200). This deviation was considered critical. Therefor experiment II was declared invalid and was repeated. Due to a technical problem of the FlowCytometer (the daily performed PQC (Performance Control) showed an Error), experiment III could not be measured, and was discontinued. This deviation was considered critical and the experiment had to be repeated.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The RFI values of the solvent control (RPMI 1640) for CD86 were 94 and 88% and for CD54 83 and 85% in Experiment I and IV, respectively, and thus the values did not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%). The cell viability was 98.84, 98.58, 99.05 and 99.00%, and thus > 90%.
- Acceptance criteria met for positive control:
The RFI values of the positive control for CD86 were 395 and 7108% and for CD54 206 and 439% in Experiment I and IV, respectively, and thus the values exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%). The cell viability was 89.85, 88.62, 87.37 and 87.01%, and thus > 50%.
- The viability of all test item concentrations were > 87.87% and thus > 50%.

Table 1: Results of Experiment I

	Concentration	Viability	Antibodies	MFI Value	MFI ratioto Isotype	RFI Value
	[µg/mL]				[%]	[%]
Medium	-	98.94%	CD86	1471	168
		98.93%	CD54	1173	134
		98.73%	ISO	878
RPMI 1640	-	98.84%	CD86	1496	160	94
		98.58%	CD54	1181	126	83
		98.19%	ISO	937
DMSO	-	98.31%	CD86	1447	158	89
		98.26%	CD54	1186	129	91
		98.85%	ISO	917	-
DNCB	4.0	89.85%	CD86	3279	-	395
		88.62%	CD54	1742	-	206
		88.73%	ISO	1188	-
Test Item	1395.4	97.64%	CD86	1524	-	89
		97.88%	CD54	1299	-	112
		97.56%	ISO	1025	-
Test Item	1674.5	98.21%	CD86	1357	-	68
		97.64%	CD54	1315	-	138
		97.48%	ISO	978	-
Test Item	2009.4	97.49%	CD86	1526	-	85
		97.53%	CD54	1406	-	146
		97.18%	ISO	1049	-
Test Item	2411.3	97.38%	CD86	1627	-	105
		96.56%	CD54	1340	-	122
		96.97%	ISO	1042	-
Test Item	2893.5	96.73%	CD86	1723	-	131
		96.94%	CD54	1400	-	168
		96.91%	ISO	990	-
Test Item	3472.2	96.72%	CD86	2078	-	206
		97.14%	CD54	1423	-	204
		96.72%	ISO	925	-
Test Item	4166.7	93.92%	CD86	2665	-	308
		93.40%	CD54	1493	-	225
		93.41%	ISO	945	-
Test Item	5000.0	93.17%	CD86	2067	-	143
		93.42%	CD54	1760	-	203
		93.31%	ISO	1265	-

Table 2: Results of Experiment IV

	Concen-tration	Viability	Antibodies	MFI Value	MFI ratioto Isotype	RFI Value
	[µg/mL]				[%]	[%]
Medium	-	98.82%	CD86	1395	182
		99.06%	CD54	1019	133
		98.90%	ISO	766
RPMI 1640	-	99.05%	CD86	1372	168	88
		99.00%	CD54	1031	126	85
		98.93%	ISO	816
DMSO	-	99.08%	CD86	1326	168	85
		98.99%	CD54	1048	132	102
		98.99%	ISO	791
DNCB	4.0	87.37%	CD86	5204		788
		87.01%	CD54	2118		439
		87.85%	ISO	990
Test Item	1395.4	98.69%	CD86	1486		100
		98.38%	CD54	1240		145
		98.80%	ISO	928
Test Item	1674.5	98.46%	CD86	1612		113
		98.47%	CD54	1309		153
		98.15%	ISO	981
Test Item	2009.4	98.37%	CD86	1582		102
		98.42%	CD54	1312		138
		98.47%	ISO	1016
Test Item	2411.3	98.06%	CD86	1737		138
		98.01%	CD54	1372		187
		98.16%	ISO	971
Test Item	2893.5	98.32%	CD86	1612		108
		98.28%	CD54	1331		148
		98.28%	ISO	1013
Test Item	3472.2	96.07%	CD86	2314		257
		95.86%	CD54	1373		228
		96.03%	ISO	883
Test Item	4166.7	91.22%	CD86	2804		351
		90.85%	CD54	1369		240
		90.24%	ISO	852
Test Item	5000.0	87.02%	CD86	2894		325
		87.38%	CD54	1620		249
		88.42%	ISO	1085

Interpretation of results:

other: skin sensitising potential based on the key event "activation of dentritic cells"

Conclusions:

Under the conditions of the test, it can be concluded, that the test substance is a sensitiser in the human Cell Line Activation Test (h-CLAT). The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.

Since 2 out of 3 key events resulted in a positive result, the test substance was considered to be a skin sensitiser. The available data meet the criteria for classification in Skin Sens. 1, H317 according to Regulation (EC) No. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

weak positive results in 2 of 3 tests

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

According to Assessment strategy for respiratory sensitisation data,

Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7a, Figure R. 7.3 -4, ECHA, 2017,

the substance is not considered for classification as respiratory sensitiser.

Justification for classification or non-classification

The available information is conclusive and sufficient for classification as Skin Sens. 1B.

The available information is conclusive and not sufficient for classification as Resp. Sens.