ε-Caprolactone, oligomers, esters with acrylic acid and 2,2,2',2'-tetrakis (hydroxymethyl)-3,3'-oxydipropan-1-ol

Endpoint summary

Description of key information

Two reliable in vitro skin sensitisation studies according to OECD 442D and OECD 442E addressing the key events inflammatory response in keratinocytes and activation of dendritic cells, respectively, and in compliance with GLP are available. Both studies showed positive reactions, thus it can be concluded that the tested substance is a skin sensitiser.

With regard to sub-categorisation, there is no evidence that ε-Caprolactone, oligomers, esters with acrylic acid and 2,2,2',2'-tetrakis (hydroxymethyl)-3,3'-oxydipropan-1-ol has the potential to produce significant sensitisation in humans and has thus to be classified as Skin Sens. Cat. 1A. This is based on the following supporting information on structural analogue substances:

- Caprolactone-Monomer (CAS 502-44-3) is described on the ECHA website as non-sensitiser according to a negative LLNA test.

- Acrylic acid and esters is a category including acrylic acid and various esters as given on the ECHA website. The category members are generally classified as Skin Sens. Cat. 1B, except for acrylic acid itself, which is not classified.

- 2,2,2',2'-tetrakis (hydroxymethyl)-3,3'-oxydipropan-1-ol (CAS 126-58-9) is also described on the ECHA website as non-sensitiser according to a negative LLNA test.

Based on those results there is no evidence that ε-Caprolactone, oligomers, esters with acrylic acid and 2,2,2',2'-tetrakis (hydroxymethyl)-3,3'-oxydipropan-1-ol has to be classified as Skin Sens. Cat. 1A. Therefore, as a worst case approach, classification as Skin Sens. Cat. 1 is determined.

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:

24 Nov - 21 Dec 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

February 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Departement Of Health Of The Government Of The United Kingdom

Type of study:

activation of keratinocytes

Details on the study design:

TEST METHOD
The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test substance using transgenic keratinocytes which were stably transfected with a selectable plasmid containing the luciferase gene under the transcriptional control of the Anti-oxidant Response Element (ARE) from a gene that is known to be up­regulated by contact sensitisers. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitiser and non-sensitisers.

TEST CELL LINE
- Type: KeratinoSens™
- Source: Givaudan, Switzerland
- Cell number at seeding: 10000 cells per well (96 well plates)
- Passage number: 14

CELL CULTURE CONDITIONS
- Type and identity of media: DMEM (Dulbecco's Modified Eagle's Medium) with GlutaMAX™ I, 1000 mg/L D-glucose, sodium pyruvate, human serum, (no geneticin used)
- Temperature (°C): 37
- CO2 (%): 5.0
- Relativ humidity: 95%

TEST CONCENTRATIONS
0.195, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, 25.0, 50.0, 100.0, 200.0 and 400.0 μg/mL

CONTROLS
Vehicle control
- Substance: dimethyl sulfoxide (DMSO)
- Final concentration: 1%

Positive control
- Substance: cinnamic aldehyde
- Final concentration: 8, 16, 32, 64 and 128 μM

EXPOSURE CONDITIONS
- Exposure duration: 48 ± 2 h

NUMBER OF REPLICATIONS: 3 runs (with 3 replicates (for luminescence and 2 for MTT))

DETERMINATION OF CELL VIABILITY
- Method: MTT assay
- Details on MTT assay design: After removal of the medium, cells were washed with HBSS. The working stock of MTT solution (1:5 dilution in DMEM from stock) was then applied to each well of the appropriate 96 well plates for 3 h prior to solubilisation using MTT solubilisation solution (50 µL). This was then mixed by shaking and read at 570 nm.

DETERMINATION OF LUMINESCENCE
- Device: luminometer
- Details on luminescence assay design: Cells were washed with PBS and then passive lysis buffer (1x; diluted from 5x using sterile water) was added, shaken for 1 min and incubated for 20 min. Following this, luciferase substrate (50 µL) was added by the luminometer and then read.

Positive control results:

The mean of the positive control cinnamic (32 µM) acid was 2.11 (fold-induction).

Key result

Run / experiment:

other: experiment 1, test substance

Parameter:

other: maximum luciferase activity induction (Imax) (mean of 3 replicates)

Value:

28.765

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: at 3.125 μg/mL

Key result

Run / experiment:

other: experiment 1, test substance

Parameter:

other: EC1.5 [µg/mL]

Remarks:

(interpolated concentration for a 1.5 fold luciferase induction)

Value:

0.36

Vehicle controls validity:

not applicable

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: experiment 2, test substance

Parameter:

other: maximum luciferase activity induction (Imax) (mean of 3 replicates)

Value:

45.011

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: at 6.25 μg/mL

Key result

Run / experiment:

other: experiment 2, test substance

Parameter:

other: EC1.5 [µg/mL]

Remarks:

(interpolated concentration for a 1.5 fold luciferase induction)

Value:

0.28

Vehicle controls validity:

not applicable

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: experiment 3, test substance

Parameter:

other: maximum luciferase activity induction (Imax) (mean of 3 replicates)

Value:

29.843

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: at 6.25 µg/mL

Key result

Run / experiment:

other: experiment 3, test substance

Parameter:

other: EC1.5 [µg/mL]

Remarks:

(interpolated concentration for a 1.5 fold luciferase induction)

Value:

0.55

Vehicle controls validity:

not applicable

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The test substance was soluble in 4% DMSO in culture medium 3 (4 x stock preparations) and min 1% DMSO in culture medium 3 (final concentrations). No precipitates were noticed in any concentration after 48 hours. An increase in metabolic activity (cell viability > 150%) was observed in cells showing a positive maximal luciferase intensity (starting at 500 μM in experiments 1 and 2).

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: The luciferase activity induced by the positive control at a concentration of 32 μM was between 1.6 and 3.0 (mean = 2.11) and the calculated EC1.5 value was between 6 and 39 μM (mean = 11.5 μM).
- Acceptance criteria met for variability between replicate measurements: The average CV of the luminescence reading for the blank values control was < 20% (mean = 13.06%).

Table 1: Results of the cytotoxicity measurement and overall induction of luciferase activity

	Concentration (µM)	Cell viability (Mean and SD)						Fold Induction (Mean and SD)
		Experiment No.						Experiment No.
		1		2		3		1		2		3
		Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Vehicle control	-	100.00	-	100.00	-	100.00	-	1.00	-	1.00	-	1.00	-
Positive control	8	99.80	7.82	114.92	6.63	110.52	1.82	1.45	0.07	2.50	0.21	1.06	0.06
	16	113.04	2.66	128.44	9.26	121.17	11.60	1.88	0.03	6.38	0.89	1.09	0.06
	32	107.62	1.34	206.35	15.66	114.38	3.07	2.84	0.22	44.71	1.94	1.39	0.05
	64	120.75	2.36	-4.85	0.77	124.36	5.21	8.58	0.43	0.00	0.00	2.67	0.31
	128	127.15	10.11	-1.04	0.36	148.96	13.64	47.44	2.45	0.00	0.00	8.73	0.69
Test substance	0.195	129.46	7.76	110.39	4.83	100.69	4.52	1.21	0.20	1.24	0.15	0.98	0.08
	0.391	115.23	2.22	107.26	3.74	98.48	1.09	1.56	0.15	1.84	0.11	1.34	0.13
	0.781	127.01	4.59	121.50	7.77	108.84	2.46	3.31	0.51	1.40	0.16	1.73	0.05
	1.563	129.60	2.24	146.27	21.64	128.19	1.38	9.19	2.05	1.97	0.15	5.59	0.59
	3.125	114.03	17.81	138.69	1.12	144.40	10.49	28.77	1.50	9.03	4.45	24.32	0.79
	6.250	-9.66	0.24	-3.15	2.47	35.23	3.92	0.06	0.04	45.01	14.42	29.84	3.04
	12.500	-4.14	0.10	-0.04	2.77	-4.71	2.10	0.00	0.00	5.29	5.27	0.02	0.00
	25.000	-14.47	0.32	-5.57	2.16	-8.15	2.06	0.01	0.00	0.01	0.00	0.00	0.00
	50.000	-14.47	0.32	-7.96	0.47	-12.01	3.31	0.00	0.00	0.01	0.00	0.01	0.00
	100.000	-9.56	3.90	-4.50	0.42	-5.58	1.23	0.00	0.00	0.01	0.00	0.00	0.00
	200.000	0.74	2.09	-0.01	0.69	-3.00	1.26	0.01	0.00	0.01	0.00	0.00	0.00
	400.000	-0.71	0.71	-1.08	2.44	-0.42	1.29	0.01	0.00	0.01	0.00	0.01	0.00

Table 2: Results of different sensitisation parameters of the test substance

	Experiment No.
	1	2	3
Imax	28.765 at 3.125 µg/mL	45.011 at 6.25 µg/mL	29.843 at 6.25µg/mL
EC1.5	0.36 µg/mL	0.28 µg/mL	0.55 µg/mL
IC50	4.743 µg/mL	5.079 µg/mL	5.827 µg/mL
IC30	4.237 µg/mL	4.638 µg/mL	5.255 µg/mL

Imax = maximal induction factor of luciferase activity compared to the solvent (negative) control measured at any test chemical concentration

EC1.5 = interpolated concentration for a 1.5 fold luciferase induction

IC50/30 = concentration effecting a reduction of cellular viability by 50/30%

 

Table 3: Results of different sensitisation parameters of the vehicle and positive control

		Experiment No.
		1		2		3
Acceptance criterion	Range	Mean	SD	Mean	SD	Mean	SD
CV% of vehicle control	< 20%	8.85	4.47	6.49	4.26	9.54	2.81
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	4	-	3	-	2	-
EC1.5 positive control	6 < x < 39	8.98	-	8	-	34.87	-
Induction positive control at 32 µM	1.6 < x < 3	2.84	0.215	44.714	1.939	1.39	0.051

CV= Coefficient of variation(a measure of variability that is calculated for a group of replicate data by dividing the standard deviation by the mean, multiplied by 100 for expression as a percentage)

Interpretation of results:

other: skin sensitising potential based on the key event “inflammatory response in keratinocytes”

Conclusions:

Under the conditions of the test, the test substance did have a keratinocyte activating potential. 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.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13 Dec 2017 - 13 Mar 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)

Version / remarks:

adopted in 2017

Deviations:

yes

Remarks:

Cytotoxicity measurement and CV75 value determination was performed by XTT test instead of flow cytometry

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of study:

activation of dendritic cells

Details on the study design:

TEST CELL LINE
- Cell type: THP-1 cells (TIB-202)
- Source: American Type Culture Collection (ATCC)
- Passage number: 24 (XTT assays), 18 and 11 (experiments 1 and 2, respectively)

CELL CULTURE CONDITIONS
- Type and identity of media: RPMI-1640, supplemented with 10% (v/v) FBS, 1% (v/v) L-glutamine, 0.05 mM 2-mercaptoethanol and 100 U/mL penicillin/ 100 μg/mL streptomycin, 4.5 g/L glucose, 1% (v/v) sodium pyruvate
- Temperature (°C): 37 ± 1.5
- CO2 (%): 5.0 ± 0.5

CONTROLS
- Negative control and vehicle control for test substance: culture medium
- Positive control: 2 and 3 μg/mL 2,4-dinitrochlorobenzene (DNCB) in DMSO (purity ≥ 99%)
- Vehicle control for positive control: 0.2% dimethyl sulfoxid (DMSO) in culture medium

PRE-EXPERIMENTS
- Solubility test: The maximum concentration of test item was 2500 µg/mL stably suspended in culture medium, as tested by a solubility test.

- Dose Finding Assay (XTT):
The XTT test is a method to investigate the cytotoxicity of a test substance. The test is based on the cleavage of the yellow tetrazolium salt, sodium 3'-(1-phenylaminocarbonyl)-(3,4 - tetrazolium)-bis-(4-methoxy-6-nitro)-benzenesulfonic acid hydrate (XTT), to form an orange water soluble formazan dye by dehydrogenase activity in active mitochondria. Two independent cytotoxicity experiments were performed on different days to obtain a reliable concentration showing 75% cell viability (CV75). The mean of two CV75 values was then used to determine the dose-range for the main experiment (h-CLAT).

After the cell seeding, 100 µL of the test item dilutions, the medium and vehicle controls, respectively, were added to the cells. All dose groups were tested in 7 replicates for each XTT test. At the end of the incubation period of 24 ± 0.5 h, the cell cultures were microscopically evaluated for morphological alterations. At the end of the incubation period, 50 µL of the XTT labelling mixture were added to each well. The cells were incubated and subsequently transferred to a microplate reader (Versamax® Molecular Devices). The absorbance was measured at 450 nm (reference wavelength 690 nm). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

A decrease in number of living cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample. This decrease directly correlates to the amount of orange formazan formed, as monitored by the absorbance.
The relative absorbance (= viability in [%]) as compared to the vehicle control is calculated using this formula:
Relative absorbance = 100 x [(mean absorbance test substance) - (absorbance blank)] / [(mean absorbance vehicle control) - (absorbance blank)]

To calculate the concentration of test substance needed to reduce the relative absorbance to 75% of the vehicle control (CV75) the following formula is used:
CV75 = Conc. > 75 - ([(Conc. > 75) - (Conc. < 75)] x [(% > 75) - 75] / [(% > 75) - (% < 75)])

a) Conc. > 75 = max. measured concentration with the % of vehicle control > 75%
b) Conc. < 75 = min. measured concentration with the % of vehicle control < 75%
c) % > 75 = relative absorbance at a) in %
d) % < 75 = relative absorbance at b) in %

Eight concentrations of the test substance were analysed. For this, dilutions were prepared by 1:2 serial dilutions from 2500 µg/mL in culture medium. Due to strong cytotoxicity observed in the first and second XTT-test, both XTT tests were repeated with adjusted test item concentrations. The highest concentration in both tests was then chosen to be 50 µg/mL (0.391, 0.781, 1.56, 3.13, 6.25, 12.5, 25.0 and 50.0 µg/mL dissolved in culture medium).
The CV75 value of the first and second XTT test: 9.2 µg/mL and 16.4 µg/mL, respectively resulting in a mean CV75 value of 12.8 µg/mL

MAIN EXPERIMENTS
The h-CLAT method is an in vitro method which quantifies changes of cell surface marker expression (CD86 and CD54) on a human cell line, THP-1 cells, following 24 ± 0.5 h exposure to 500 µL of the test substance. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorescein isothiocyanate (FITC)-labelled antibodies for 30 ± 5 min. The relative fluorescence intensity of the surface markers compared to the vehicle control are calculated and used in the prediction model to support the discrimination between sensitisers and non-sensitisers. Dead cells were determined by staining with 7-AAD (7-amino-actinomycin D).

EXPOSURE CONDITIONS
- Exposure duration: 24 ± 0.5 h

TEST CONCENTRATIONS
- Justification for top dose: Eight final concentrations (4.3, 5.2, 6.2, 7.4, 8.9, 10.7, 12.8 and 15.4 µg/mL) were used for the test substance in the main experiment (h-CLAT). The highest concentration used was 1.2 × CV75 and seven further concentrations of the test item were prepared by serial 1:1.2 dilution.

NUMBER OF REPLICATIONS: 2 independent experiments

STAINING
- Antibodies: FITC labelled anti-CD54, FITC anti-CD86, FITC mouse IgG1 (isotype control)
- Temperature: 2 - 8 °C (on ice)
- Duration: 30 ± 5 min in the dark

MEASUREMENT
- Device: FACS (FACSCalibur, Becton Dickinson GmbH)
- Software: Cellquest Pro 6.0

EVALUATION CRITERIA
Calculation:
The relative fluorescence intensity (RFI) is used as an indicator of CD86 and CD54 expression, and is calculated for each concentration as follows:

RFI (%) = [(MFI of test substance treated cells) - (MFI of test substance treated isotype control cells) / (MFI of vehicle treated cells) - (MFI of vehicle treated isotype control cells)] x 100
MFI: geometric mean fluorescence intensity (GeoMean)

The cell viability is calculated as follows for each concentration:
Cell viability (%) = (Mean cytotox of vehicle treated cells) / (Mean cytotox of test substance treated cells) x 100
Mean cytotox is the mean of GeoMean(7-AAD) isotype control, GeoMean(7-AAD) CD54 and GeoMean(7-AAD) CD86.

Prediction Model:
The test substance is tested in at least 2 independent runs.
The test substance is considered to be a sensitizer, if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs:
- the RFI of CD86 is ≥ 150% at any tested dose at a cell viability ≥ 50%
- the RFI of CD54 was ≥ 200% at any tested dose at a cell viability ≥ 50%
Otherwise it is considered to be a non-sensitizer.

Based on the above, different results in both runs would require the performance of a third run. In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 (hereinafter refered to as P1) and the other is only positive for CD54 (hereinafter refered to as P2), a third run is required. If this third run is negative for both markers (hereinafter refered to as N), the h-CLAT prediction is considered negative. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter refered to as P12), the h-CLAT prediction is considered positive. An h-CLAT prediction should be considered in the framework of an IATA (OECD 442E guideline).

ACCEPTANCE CRITERIA
The study is considered as valid, if the following criteria are met:
- Cell viability of vehicle control is > 90%
- The cell viability of at least 4 doses of the test substance in each experiment is ≥ 50%.
- In the positive control, RFI values of both CD86 and CD54 is ≥ 150% and ≥ 200%, respectively at a cell viability of > 50% in at least one concentration of the two tested positive control concentrations
- In the vehicle control, RFI values of both CD86 and CD54 should be < 150% and < 200%, respectively
- The MFI ratio of CD86 and CD54 to isotype control IgG1 control for the medium and DMSO control is > 105%.
Negative results are acceptable only for test items exhibiting a cell viability of < 90% at the highest concentration tested (i.e. 1.2 × CV75). If the cell viability at 1.2 × CV75 is ≥ 90% the negative result should be discarded. In such case it is recommended to try to refine the dose selection by repeating the CV75 determination. It should be noted that when 5000 μg/mL in saline (or medium or other solvents/vehicles), 1000 μg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test chemical, a negative result is acceptable even if the cell viability > 90% (OECD 442E guideline).

Positive control results:

The positive control led to upregulation of CD86 (≥ 150% in both experiments and for both concentrations tested (2 and 3 µg/mL)) and CD54 (≥ 200% in both experiments and for both concentrations tested) while the cell viability was > 50%.

Key result

Run / experiment:

other: 24 h incubation (experiment 1)

Parameter:

other: relative fluorescence intensity (RFI) of CD54 (%)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

RFI CD54 was < 200% or the cell viability was < 50%

Key result

Run / experiment:

other: 24 h incubation (experiment 2)

Parameter:

other: relative fluorescence intensity (RFI) of CD54 (%)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

exceeding positive criteria only at 15.4 μg/mL (250.9%)

Key result

Run / experiment:

other: 24 h incubation (experiment 1)

Parameter:

other: relative fluorescence intensity (RFI) of CD86 (%)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

exceeding positive criteria, except of at 4.3 and 6.2 μg/mL (RFI CD86 < 150%) and at 12.8 and 15.4 μg/mL (RFI CD86 > 150%, but cell viability <50%)

Key result

Run / experiment:

other: 24 h incubation (experiment 2)

Parameter:

other: relative fluorescence intensity (RFI) of CD86 (%)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

exceeding positive criteria, except of at 4.3 μg/mL (RFI CD86 < 150%)

Other effects / acceptance of results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxic effects (i.e. cell viability less than 50%) were observed following incubation with the test substance at concentrations greater or equal to 12.8% in experiment 1. No cytotoxic effects were observed at any concentration tested in experiment 2.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
no information given in the report

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control:
yes, as the respective cell viabilities were > 90% (97 and 100% in experiment 1 and 2, respectively) and the respective RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 ≥ 150% (99.5 and 103.6% in experiment 1 and 2, respectively) and CD54 ≥ 200% (87.3 and 125.9% in experiment 1 and 2, respectively).

- Acceptance criteria met for positive control:
yes, as the respective RFI values of both CD86 and CD54 exceeded the positive criteria (CD54 ≥ 200% ( 240.6/ 268.8% and 249.3/ 350.7 for 2/ 3 µg/mL in experiment 1 and 2, respectively) and CD86 ≥ 150% (661.6/ 772.6% and 525.3/ 787.4% for 2/ 3 µg/mL in experiment 1 and 2, respectively)) and the cell viability was > 50%.

- For both medium and vehicle controls, the MFI ratio of both CD86 and CD54 to isotype control was > 105% (205.8% and 215.5% (medium) and 205.8% and 228.4% (DMSO) for CD86 in experiment 1 and 2, respectively and 152.9% and 145.4% (medium) and 146.4% and 161.3% (DMSO) for CD54 in experiment 1 and 2, respectively, thus meeting this criteria.

- For the test substance, the cell viability was be more than 50% in at least 4 tested concentrations in each experiment (6/8 concentrations in experiment 1, all 8 concentrations in experiment 2), thus meeting this criteria.

Table 1: Results of the first XTT test

	Concentration [µg/mL]	Microscopic evaluation	Photometric evaluation
		Cytotoxicity ***	Mean absorbance*^§	SD	Blank^§	Mean absorbance – Blank	Absorbance in % of vehicle control**
Medium control	-	no	0.798	0.034	0.235	0.563	95.21
Vehicle control	-	no	0.825	0.031	0.234	0.592	100.00
Test substance	0.391	no	0.808	0.036	0.236	0.572	96.74
	0.781	no	0.831	0.033	0.234	0.596	100.82
	1.56	no	0.866	0.047	0.231	0.634	107.21
	3.13	no	0.902	0.038	0.232	0.670	113.18
	6.25	no	0.818	0.055	0.235	0.583	98.59
	12.5	yes	0.515	0.070	0.232	0.283	47.86
	25	yes	0.262	0.038	0.234	0.028	4.69
	50	yes	0.240	0.007	0.231	0.009	1.53

§ = Given absorbances are rounded values

* = mean absorbance (absolute) of 7 wells

** = relative absorbance [rounded values]

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

 

The mean viability of the solvent control in comparison to the medium control was 105.03%.

The CV75 value of the first XTT test: 9.2 µg/mL

Table 2: Results of the second XTT test

	Concentration [µg/mL]	Microscopic evaluation	Photometric evaluation
		Cytotoxicity ***	Mean absorbance*^§	SD	Blank^§	Mean absorbance – Blank	Absorbance in % of vehicle control**
Medium control	-	no	0.862	0.031	0.231	0.63	89.83
Vehicle control	-	no	0.937	0.114	0.235	0.702	100.00
Test substance	0.391	no	1.109	0.237	0.231	0.878	125.11
	0.781	no	1.018	0.212	0.223	0.796	113.39
	1.56	no	0.963	0.115	0.244	0.720	102.53
	3.13	no	1.035	0.182	0.238	0.797	113.49
	6.25	no	1.076	0.212	0.236	0.840	119.72
	12.5	no	0.890	0.033	0.238	0.651	92.81
	25	yes	0.483	0.046	0.231	0.252	35.91
	50	yes	0.389	0.052	0.239	0.151	21.49

§ = Given absorbances are rounded values

* = mean absorbance (absolute) of 7 wells

** = relative absorbance [rounded values]

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

 

The mean viability of the solvent control in comparison to the medium control was 111.32%.

The CV75 value of the first XTT test: 16.4 µg/mL

The mean CV75 value of both XTT tests: 12.8 µg/mL

Table 3. Results of the first h-CLAT run

	Concentration (µg/mL)	Antibody / ISO	MFI GeoMean (FITC)	MFI - ISO	RFI (%)	Cyto(Geo) GeoMean (7-AAD)	Mean Cyto(Geo) GeoMean (7-AAD)	Cell viability (%)
Medium control	-	ISO	2.08	-	-	3.47	3.3	100.0
		CD54	3.18	1.10	100.0	3.34
		CD86	4.28	2.20	100.0	3.01
DMSO control	-	ISO	2.07	-	-	3.59	3.2	100.0
		CD54	3.03	0.96	100.0	3.29
		CD86	4.26	2.19	100.0	2.82
Positive control (DNCB)	2.0	ISO	2.91	-	-	6.25	4.9	66.0
		CD54	5.22	2.31	240.6 *	5.53
		CD86	17.40	14.49	661.6 *	2.91
	3.0	ISO	2.98	-	-	6.31	5	64.1
		CD54	5.56	2.58	268.8 *	5.75
		CD86	19.90	16.92	772.6 *	3.08
Test Item	4.3	ISO	2.27	-	-	4.02	3.6	91.3
		CD54	3.31	1.04	94.5	3.59
		CD86	5.08	2.81	127.7	3.15
	5.2	ISO	2.38	-	-	4.20	3.7	87.8
		CD54	3.53	1.15	104.5	3.82
		CD86	5.84	3.46	157.3 *	3.16
	6.2	ISO	2.38	-	-	4.31	4.0	82.6
		CD54	3.59	1.21	110.0	3.94
		CD86	5.26	2.88	130.9	3.64
	7.4	ISO	2.60	-	-	4.98	4.2	77.3
		CD54	3.93	1.33	120.9	4.32
		CD86	7.77	5.17	235.0 *	3.40
	8.9	ISO	2.81	-	-	5.53	4.7	69.3
		CD54	4.35	1.54	140.0	5.00
		CD86	9.22	6.41	291.4 *	3.64
	10.7	ISO	3.30	-	-	7.26	6.2	52.6
		CD54	5.33	2.03	184.5	6.77
		CD86	14.08	10.78	490.0 *	4.65
	12.8	ISO	3.67	-	-	8.63	7.5	43.9
		CD54	5.67	2.00	181.8	8.21
		CD86	14.63	10.96	498.2 *	5.53
	15.4	ISO	4.16	-	-	10.34	8.9	36.9
		CD54	6.59	2.43	220.9 *	9.47
		CD86	22.43	18.27	830.5 *	6.83

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

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

Table 4. Results of the second h-CLAT run

	Concentration (µg/mL)	Antibody / ISO	MFI GeoMean (FITC)	MFI - ISO	RFI (%)	Cyto(Geo) GeoMean (7-AAD)	Mean Cyto(Geo) GeoMean (7-AAD)	Cell viability (%)
Medium control	-	ISO	2.38	-	-	4.09	3.7	100.0
		CD54	3.46	1.08	100.0	3.79
		CD86	5.13	2.75	100.0	3.33
DMSO control	-	ISO	2.22	-	-	4.05	3.7	100.0
		CD54	3.58	1.36	100.0	3.72
		CD86	5.07	2.85	100.0	3.18
Positive control (DNCB)	2.0	ISO	2.61	-	-	5.77	4.5	81.2
		CD54	6.00	3.39	249.3 *	4.75
		CD86	17.58	14.97	525.3 *	2.97
	3.0	ISO	2.73	-	-	6.64	4.9	74.3
		CD54	7.50	4.77	350.7 *	5.19
		CD86	25.17	22.44	787.4 *	2.91
Test Item	4.3	ISO	2.39	-	-	4.43	4	93.8
		CD54	3.68	1.29	119.4	3.99
		CD86	5.85	3.46	125.8	3.53
	5.2	ISO	2.43	-	-	4.20	3.8	99.1
		CD54	3.90	1.47	136.1	3.80
		CD86	6.93	4.50	163.6 *	3.31
	6.2	ISO	2.50	-	-	4.40	3.9	96.7
		CD54	3.96	1.46	135.2	3.90
		CD86	6.93	4.43	161.1 *	3.29
	7.4	ISO	2.54	-	-	4.78	4.1	91.4
		CD54	3.87	1.33	123.1	4.28
		CD86	8.22	5.68	206.5 *	3.20
	8.9	ISO	2.32	-	-	4.21	3.8	98.9
		CD54	3.84	1.52	140.7	3.95
		CD86	7.37	5.05	183.6 *	3.18
	10.7	ISO	2.61	-	-	5.33	4.5	82.4
		CD54	4.34	1.73	160.2	4.58
		CD86	8.16	5.55	201.8	3.69
	12.8	ISO	2.68	-	-	5.10	4.1	90.1
		CD54	4.35	1.67	154.6	4.42
		CD86	10.39	7.71	280.4 *	2.92
	15.4	ISO	3.29	-	-	7.52	5.7	65.6
		CD54	6.00	2.71	250.9 *	6.07
		CD86	16.84	13.55	492.7 *	3.51

* = RFI value of CD86 or CD54 fulfilled the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%

Table 5: Historical Control Data (2015/2016)

	Positive Control DNCB				DMSO- /Medium Control
	2 µg/mL		3 µg/mL		(MFI - MFI(ISO) DMSO)/ (MFI - MFI(ISO) Medium)
Antibody	CD54	CD86	CD54	CD86	CD54	CD86
Mean value [%]	466.9	373.2	655.1	437.4	112.7	103.5
SD	248.3	138.4	268.4	162.2	16.3	17.4
Max value [%]	1714.1	837.1	1492.2	950.5	151.5	144.9
Low value [%]	200.8	200	266.7	185.8	62.6	74.8

RFI value of CD86 or CD54 fulfills the positive criteria if CD86 ≥ 150% and CD54 ≥ 200%.

Interpretation of results:

other: skin sensitising potential based on the key event “activation of dendritic cells”

Conclusions:

Under the conditions of the test, the test substance induces dendritic cell activation. 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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on skin sensitsation meet the criteria for classification according to Regulation (EC) 1272/2008, and is therefore classified as Skin Sens. Category 1 (H317).