Reaction mass of rel-(2S,4R,6S)-2,4,6-trimethyl-4-phenyl-1,3-dioxane and rel-(2S,4S,6S)-2,4,6-trimethyl-4-phenyl-1,3-dioxane

Endpoint summary

Description of key information

Skin sensitisation (OECD 442C and 442D): negative

Skin sensitisation (OECD 442E): positive

WoE conclusion from in vitro skin sensitisation battery: not skin sensitising

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:

09 -18 Mar 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

yes

Remarks:

No information on stability, precision and co-elution controls provided. No information on stability/solubility of test substance after preparation of peptide depletion samples and after 22 h incubation.

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM, UK

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

TEST METHOD
The DPRA is an in chemico test system proposed to address the molecular initiating event of the skin sensitisation adverse outcome pathway, namely protein reactivity, by substance towards model synthetic peptides containing either lysine or cysteine. The DPRA quantifies the free concentration of cysteine- or lysine-containing peptide following incubation with the test substance. Relative peptide concentration is measured by HPLC with gradient elution and UV detection at 220 nm. Cysteine- and lysine peptide percent depletion values are then calculated and used in the prediction model which allows assigning the test substance to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

TEST SYSTEM
- Supplier: AnaSpec
- Synthetic cysteine-containing peptide:
Alternative name: Ac-RFAACAA-OH
Batch number: 1556171
Molecular weight: 751.5 g/L
Purity: 95%
Expiry date: 5 years
- Synthetic lysine-containing peptide:
Alternative name: Ac-RFAAKAA-OH
Batch number: 1556172
Molecular weight: 776 g/L
Purity: 90 - 95%
Expiry date: 5 years

SOLVENT CONTROL AND ASSESSMENT OF TEST ITEM SOLUBILITY
- Solvent: acetonitrile
The solubility of the test item in acetonitrile was assessed at a concentration of 100 mM.

POSITIVE CONTROL
- Substance: cinnamic aldehyde
- Batch number: MKBR2427V
- Purity: >95%
- Expiry date: Feb 2019
The positive control was prepared at a concentration of 100 mM.

STABILITY AND PRECISION CONTROL
Stability and precision controls of both peptides were prepared at a concentration of 0.5 mM.

PEPTIDE STOCK SOLUTION PREPARATION
Cysteine-containing peptide:
- Solvent: phosphate buffer (pH 7.5)
- Concentration: 0.667 mM
Lysine-containing peptide:
- Solvent: ammonium acetate buffer (pH 10.2)
- Concentration: 0.667 mM

INCUBATION CONDITIONS OF THE TEST SUBSTANCE WITH THE PEPTIDE SOLUTIONS
- Peptide to test substance ratios: Cysteine-containing peptide: 1:10 (0.5 mM peptide, 5 mM test substance/positive control); Lysine-containing peptide: 1:50 (0.5 mM peptide, 25 mM test substance/positive control)
- Temperature used during treatment / exposure: 25 °C
- Duration of treatment / exposure: at least 22 h prior to initiation of the analysis run

NUMBER OF REPLICATES
for each peptide in triplicates for treatment and control

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Waters Alliance 2695 separation module and 2487 dual wavelength detector
- Column: Agilent Zorbax SB C18, 3.5 µm, 100 x 2.1 mm with guard column Phenomenex AJO4286
- HPLC mobile phase:
A: 0.1% (v/v) trifluoracetic acid in deionised water
B: 0.085% (v/v) trifluoracetic acid in acetonitrile
- Flow: 0.35 mL/min
- Gradient:
Time (min): 0, 20, 21, 23, 23.5, 30
% B: 10, 25, 90, 90, 10, 10
- Detector Wavelength: 220 nm
- Calibration standard concentrations of both peptides: 0.0167, 0.0334, 0.0667, 0.133, 0.267 and 0.534 mM
- Column temperature: 30 °C

Key result

Run / experiment:

other: ≥ 22 h incubation

Parameter:

other: % depletion of cysteine-containing peptide

Remarks:

(mean value of 3 replicates)

Value:

0.906 %

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Key result

Run / experiment:

other: ≥ 22 h incubation

Parameter:

other: % depletion of lysine-containing peptide

Remarks:

(mean value of 3 replicates)

Value:

-1.68 %

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Key result

Run / experiment:

other: ≥ 22 h incubation

Parameter:

other: % overall mean depletion

Value:

0.453 %

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Other effects / acceptance of results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: No co-elution of the test substance occurred during the assay. The solubility of the test substance in acetonitrile at a nominal concentration of 100 mM was confirmed.

ACCEPTANCE OF RESULTS:
All analytical acceptance criteria for each peptide were met.

Table 5. Mean peptide depletion of cysteine-containing peptide.

	Peak area (µV.sec)	Peptide concentration (µg/mL)*	Peptide depletion (%)**	Mean depletion ± CV (%)
Test substance	902257	381	0.608	0.906 ± 0.28
	899025	379	0.964
	897389	378	01.15
Positive control	244716	103	73.0	73.0 ± 0.35
	245752	104	72.9
	244042	103	73.1

*: Samples prepared at a concentration of 376 μg/mL (0.5 mM)

**: Calculated against a mean reference control peak area of 940000 μV.sec (n=6)

Table 6. Mean peptide depletion of lysine-containing peptide.

	Peak area (µV.sec)	Peptide concentration (µg/mL)*	Peptide depletion (%)**	Mean depletion ± CV (%)
Test substance	792447	410	-3.34	-1.68 ± 1.81
	782216	405	-2.00
	764584	396	0.295
Positive control	337396	175	56.0	56.3 ± 1.54
	338682	176	55.8
	329160	171	57.1

*: Samples prepared at a concentration of 388 μg/mL (0.5 mM)

**: Calculated against a mean control peak area of 766850 μV.sec (n=6)

Interpretation of results:

other: DPRA prediction: negative (no or minimal reactivity) according to OECD 442C

Conclusions:

Under the conditions of the Direct Peptide Reactivity Assay the test substance showed no or minimal peptide reactivity.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

5 Sept - 10 Oct 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 4 Feb 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

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 by addressing 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 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
- Passage number: 9 (Experiment 1), 12 (Experiment 2)

CELL CULTURE CONDITIONS
- Type and identity of media:
Maintenance medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) supplemented with 1.0 g/L D-glucose and Na-pyruvate, 10% fetal bovine calf serum and 1% geneticin (final concentration 500 µg/mL)
Assay medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) supplemented with 1.0 g/L D-glucose and Na-pyruvate and 10% fetal bovine calf serum
Test substance exposure medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) supplemented with 1.0 g/L D-glucose and Na-Pyruvate and 1% fetal bovine calf serum
- Temperature (°C): 37 ± 1.0
- CO2 (%): 5.0

TEST CONCENTRATIONS
0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 61.5, 125, 250, 500, 1000 and 2000 µM

CONTROLS
Solvent control:
- Substance: dimethyl sulfoxide (DMSO)
- Final concentration: 1% (v/v)
Positive control:
- Substance: cinnamic aldehyde
- Final concentration: 4 - 64 µM

EXPOSURE CONDITIONS
- Exposure duration: 48 ± 1 h

NUMBER OF REPLICATIONS: triplicates (test substance and positive control) or sextuplicates (solvent control) in two independent experiments

DETERMINATION OF CELL VIABILITY
- Method: MTT assay
- MTT concentration: 5 mg/mL
- Incubation time: 4 h
- Device: plate reader
- Wavelength: 600 nm

DETERMINATION OF LUMINESCENCE
- Cell lysis reagent: Luciferase Cell Culture Lysis 5x Reagent Kit (Promega, Cat. No. E1531, Lot No. 0000199324)
- Device: plate reader

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: maximum luciferase activity induction (Imax)

Value:

0.98

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: test item concentration: 1000 µM; cell viability: 105.1%

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: maximum luciferase activity induction (Imax)

Value:

1.22

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: test item concentration: 250 and 500 µM; cell viability: 111.4 and 109.7%

Other effects / acceptance of results:

ADDITIONAL INFORMATION ON CYTOTOXICITY: Clear evidence of cytotoxicity (cell viability < 70%) was observed at 2000 µM.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: The average coefficient of variation of the luminescence reading for the vehicle control was < 20% (7.6% in and 10.5% in Experiment 1 and 2, respectively).
- Acceptance criteria met for positive control: The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (4.61 and 2.12 in Experiment 1 and 2, respectively) and the calculated EC1.5 value was between 7 and 30 µM (18.81 and 27.06 µM in Experiment 1 and 2, respectively).

Table1. Results of the cytotoxicity measurement

	Concentration [µM]	Cell Viability (%)
		Experiment 1	Experiment 2	Mean ± SD
Vehicle control	-	100	100	100 ± 0.0
Positive control	4	98.5	98.8	98.7 ± 0.2
	8	106.0	109.0	107.5 ± 2.1
	16	111.3	110.2	110.7 ± 0.8
	32	122.4	111.3	116.9 ± 7.8
	64	126.8	125.1	125.9 ± 1.2
Test substance	0.98	102.6	106.1	104.4 ± 2.5
	1.95	95.6	98.8	97.2 ± 2.3
	3.91	98.7	100.3	99.5 ± 1.1
	7.81	95.2	90.4	92.8 ± 3.4
	15.63	94.6	101.4	98.0 ± 4.7
	31.25	90.3	109.5	99.5 ± 1.1
	62.50	90.6	106.1	92.8 ± 3.4
	125	92.9	107.3	100.1 ± 10.2
	250	92.9	111.4	102.1 ± 13.1
	500	92.7	109.7	101.2 ± 12.0
	1000	105.1	111.8	108.4 ± 4.7
	2000	0.1	-0.1	0.0 ± 0.1

Table 2. Induction of luciferase activity

	Concentration [µM]	Fold induction
		Experiment 1	Experiment 2	Mean ± SD
Vehicle control	-	1.00	1.00	1.00 ± 0.00
Positive control	4	1.08	1.10	1.09 ± 0.01
	8	1.26	1.24	1.25 ± 0.02
	16	1.31	1.29	1.30 ± 0.01
	32	2.37	1.59	1.98 ± 0.55
	64	4.61	2.12	3.37 ± 1.76
Test substance	0.98	0.86	1.01	0.93 ± 0.11
	1.95	0.86	1.13	1.00 ± 0.19
	3.91	0.92	1.16	1.04 ± 0.17
	7.81	0.88	1.13	1.01 ± 0.18
	15.63	0.93	1.09	1.01 ± 0.12
	31.25	0.84	1.08	0.96 ± 0.16
	62.50	0.91	1.16	1.04 ± 0.18
	125	0.91	1.21	1.06 ± 0.21
	250	0.89	1.22	1.05 ± 0.24
	500	0.91	1.22	1.06 ± 0.22
	1000	0.98	1.14	1.06 ± 0.11
	2000	0.00	0.00	0.00 ± 0.00

Interpretation of results:

other: activation of keratinocytes negativ according to OECD 442D

Conclusions:

Under the conditions of the ARE-Nrf2 Luciferase test the test substance did not induce luciferase activity in the transgenic KeratinoSens™ cell line.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

07 Mar - 10 Nov 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

December 2015

Deviations:

yes

Remarks:

Cytotoxicity measurement and estimation of the CV75 value 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
- Strain: THP-1 cells (human monocytic leukemia cell line)
- Source: American Type Culture Collection
- Passage number: 5 and 7 (XTT test); 9, 10 and 21 (h-CLAT)

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

DOSE FINDING ASSAY BY CYTOTOXICITY MEASUREMENT (XTT TEST)
The doses investigated in the main experiment (h-CLAT) were determined with a XTT test. 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 with different cell cultures or on different days to obtain a reliable concentration showing 75% cell viability (CV75).

CONTROLS
Negative control
- Substance: culture medium
Solvent control
- Substance: dimethyl sulfoxide (DMSO)
- Concentration: 0.2 - 0.5%

EXPOSURE CONDITIONS
- Exposure duration: 24 ± 1 h

TEST CONCENTRATIONS
- Justification for top concentration: The test substance was soluble in DMSO up to and including 250 µg/mL as tested by a solubility test.
1.91, 3.83, 7.66, 15.32, 30.64, 61.28, 122.55, 245.10 µg/mL (XTT 1); 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125 and 250 µg/mL (XTT 2)

NUMBER OF REPLICATIONS: quadruplicates per concentration in two independent experiments

MEASUREMENT
- Device: microplate reader (Versamax Molecular Devices)
- Wavelength: 450 nm
- Reference wavelength: 690 nm

EVALUATION CRITERIA
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 as compared to the solvent control is calculated using the formula:
Relative absorbance = 100 x [(mean absorbance test substance) - (absorbance blank)] / [(mean absorbance solvent control) - (absorbance blank)]

To calculate the concentration of test substance needed to reduce the relative absorbance to 75% of the solvent 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 solvent control > 75%
b) Conc. < 75: min. measured concentration with the % of solvent control < 75%
c) % > 75: relative absorbance at a) in %
d) % < 75: relative absorbance at b) in %

ACCEPTANCE CRITERIA
The XTT test is considered to be acceptable if it meets the following criteria:
- mean absorbance of the negative control is ≥ 0.5
- mean viability of the solvent control is ≥ 70%

h-CLAT (main study)
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 h exposure to the test substance. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorescein isothiocyanate (FITC)-labelled antibodies. The relative fluorescence intensity of surface markers compared to solvent control are calculated and used in the prediction model to support the discrimination between sensitisers and non-sensitisers.

CONTROLS
Negative Control
- Substance: culture medium
Solvent control
- Substance: dimethyl sulfoxide (DMSO)
- Concentration: 0.2%
Positive control
- Substance: 2,4-dinitrochlorobenzene (DNCB) in DMSO
- Concentration: 2 and 3 µg/mL

EXPOSURE CONDITIONS
- Exposure duration: 24 ± 25 min

TEST CONCENTRATIONS
- Justification for top dose: Due to a lack in toxicity in the XTT test, a CV75 could not be determined. Therefore, the highest soluble concentration (250 µg/mL) multiplied with 1.2 was prepared as highest concentration.
84, 100, 121, 145, 174, 208, 250 and 300 µg/mL

NUMBER OF REPLICATIONS: at least in triplicates for the different stainings in three independent experiments

STAINING
- Antibodies: FITC anti-CD54 (Clone: Fun-1); FITC anti-CD86 (Clone: 6.5B5); FITC mouse IgG1 (isotype control)
- Temperature: 2 - 8 °C
- Duration: 30 ± 5 min

MEASUREMENT
- Device: flow cytometer FACSCalibur (Becton Dickinson)

EVALUATION CRITERIA
The relative fluorescence intensity (RFI) is used as an indicator of CD86 and CD54 expression, and is calculated as follows for each concentration:
RFI (%) = [(MFI of test substance treated cells) - (MFI of test substance isotype control cells) / (MFI of solvent control cells) - (MFI of solvent 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 solvent control cells) / (mean cytotox of test substance treated cells) x 100
Where the mean cytotox is the mean of GeoMean (7-AAD) isotype control, GeoMean (7-AAD) CD54 and GeoMean (7-AAD) CD86.

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

ACCEPTANCE CRITERIA
The study is considered as valid, if the following criteria are met:
- Cell viability of negative control is adjusted to 100% and the cell viability of the solvent control should be more than 90% in comparison to the negative control.
- In the positive control, RFI values of both CD86 and CD54 should exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50%.
- In the vehicle control, RFI values compared to the negative control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
- For negative and solvent controls, the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
- For the test substance resulting in negative outcome, the cell viability at the 1.2 × CV75 value should be less than 90%. If the cell viability at the 1.2 × CV75 value is more than 90% for a positive tested test substance, the data will be acceptable. If 5 mg/mL in saline, 1 mg/mL in DMSO or the highest soluble dose will be used as the maximal test concentration instead of CV75-based dose, the data for test substance are accepted independent by the cell viability.
- The cell viability of at least 4 doses in each experiment should be ≥50%.

Run / experiment:

other: 24 h incubation

Parameter:

other: relative fluorescence intensity of CD86 (%)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: in 2 out of 3 independent run data

Run / experiment:

other: 24 h incubation

Parameter:

other: relative fluorescence intensity of CD54 (%)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: in 2 out of 3 independent run data

Other effects / acceptance of results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
XTT test
Cytotoxic effects were not observed following incubation with the test item up to the highest soluble concentration (250 µg/mL). The viability of the cells in both XTT spaced within a range of 92.67 to 112.4% and 81.35 to 108.61%, respectively. Due to the lack of cytotoxicity, a CV75 value could not be calculated.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for solvent control: In the solvent control, RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and did not induce cytotoxic effects.
- Acceptance criteria met for positive control: The RFI values of the positive control for CD86 and CD54 exceeded the positive criteria (CD86 > 150% and CD54 > 200%) and the cell viability was > 50%.

Table 1. Results of the first XTT Test

	Concentration (µg/mL) #	Mean absorbance*	SD	Blank	Absorbance in % of vehicle control**
Negative control		0.741	0.056	0.249	114.31
Vehicle Control		0.677	0.040	0.246	100.00
Test substance	1.91	0.690	0.028	0.242	103.92
	3.83	0.675	0.026	0.244	99.99
	7.66	0.702	0.019	0.241	106.98
	15.32	0.727	0.082	0.243	112.40
	30.64	0.721	0.121	0.246	110.26
	61.28	0.691	0.051	0.248	102.78
	122.55	0.645	0.046	0.245	92.73
	245.10	0.644	0.026	0.245	92.67

*: mean absorbance (absolute) of 4 wells

**: relative absorbance

#: The test item was formulated without adjustment to purity in the first dose finding assay (XTT Test). Therefore, the used test item concentrations were corrected to the purity of the test item with a correction factor of 1.02.

 

Table 2. Results of the second XTT Test

	Concentration (µg/mL)	Mean absorbance*	SD	Blank	Absorbance in % of vehicle control**
Negative control		0.759	0.011	0.278	109.97
Vehicle Control		0.718	0.054	0.281	100.00
Test substance	1.95	0.715	0.035	0.277	100.18
	3.91	0.705	0.049	0.279	97.30
	7.81	0.709	0.105	0.274	99.26
	15.63	0.651	0.054	0.272	86.59
	31.25	0.634	0.042	0.278	81.35
	62.5	0.677	0.042	0.280	90.78
	125	0.719	0.035	0.278	100.82
	250	0.756	0.051	0.281	108.61

*: mean absorbance (absolute) of 4 wells

**: relative absorbance

Table 3. Results of the first h-CLAT run

	Concentration (µg/mL)	Antibody / ISO	MFI GeoMean(FITC)	MFI-ISO	Cyto(Geo) GeoMean(7-AAD)	Mean Cyto	RFI (%)	Viability (%)
Negative control	-	ISO	2.11		1.94	1.8		100.0
		CD54	2.58	0.47	2.12		100.0
		CD86	4.19	2.08	1.41		100.0
Vehicle control	-	ISO	2.07		1.67	1.7		100.0
		CD54	2.69	0.62	2.00		100.0
		CD86	4.47	2.40	1.40		100.0
Positive control	2	ISO	2.38		4.41	3.2		52.8
		CD54	5.11	2.73	2.86		440.3*
		CD86	7.55	5.17	2.34		215.4*
	3	ISO	2.35		3.24	2.6		65.0
		CD54	7.22	4.87	2.30		785.5*
		CD86	6.81	4.46	2.26		185.8*
Test substance	84	ISO	2.01		2.11	2.5		66.8
		CD54	2.60	0.59	2.96		95.2
		CD86	3.25	1.24	2.52		51.7
	100	ISO	2.05		2.01	2.3		75.0
		CD54	2.65	0.60	2.62		96.8
		CD86	3.67	1.62	2.13		67.5
	121	ISO	1.97		3.38	2.9		57.9
		CD54	2.73	0.76	2.43		122.6
		CD86	3.65	1.68	2.95		70.0
	145	ISO	1.98		3.85	2.5		69.0
		CD54	2.76	0.78	1.94		125.8
		CD86	3.53	1.55	1.56		64.6
	174	ISO	2.05		2.82	2.1		81.8
		CD54	2.81	0.76	1.87		122.6
		CD86	3.74	1.69	1.51		70.4
	208	ISO	2.04		2.68	2.1		82.4
		CD54	2.82	0.78	2.00		125.8
		CD86	3.51	1.47	1.47		61.3
	250	ISO	2.18		2.75	2.3		72.8
		CD54	2.95	0.77	2.70		124.2
		CD86	3.82	1.64	1.51		68.3
	300^PS	ISO	2.88		2.29	2.6		64.7
		CD54	10.55	7.67	2.93		1237.1*
		CD86	6.30	3.42	2.62		142.5

^PSPhase separation (oily droplets)

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

 

Table 4. Results of the second h-CLAT run

	Concentration (µg/mL)	Antibody / ISO	MFI GeoMean(FITC)	MFI-ISO	Cyto(Geo) GeoMean(7-AAD)	Mean Cyto	RFI (%)	Viability (%)
Negative control	-	ISO	2.02		2.07	1.9		100.0
		CD54	2.78	0.76	1.88		100.0
		CD86	4.04	2.02	1.77		100.0
Vehicle control	-	ISO	2.32		1.92	2.0		100.0
		CD54	2.96	0.64	2.24		100.0
		CD86	3.83	1.51	1.77		100.0
Positive control	2	ISO	2.33		3.06	2.0		99.0
		CD54	13.30	10.97	1.48		1714.1*
		CD86	10.71	8.38	1.45		555.0*
	3	ISO	2.31		4.16	3.3		59.9
		CD54	11.86	9.55	3.01		1492.2*
		CD86	7.70	5.39	2.73		357.0*
Test substance	84	ISO	2.00		2.44	2.2		90.8
		CD54	2.93	0.93	2.07		145.3
		CD86	3.57	1.57	2.02		104.0
	100	ISO	2.01		2.60	2.3		85.7
		CD54	3.02	1.01	2.51		157.8
		CD86	3.74	1.73	1.81		114.6
	121	ISO	2.06		2.52	2.1		94.9
		CD54	3.19	1.13	2.06		176.6
		CD86	4.58	2.52	1.67		166.9*
	145	ISO	2.02		2.67	2.3		84.5
		CD54	3.02	1.00	2.42		156.3
		CD86	3.76	1.74	1.93		115.2
	174	ISO	2.03		2.64	2.7		73.8
		CD54	3.10	1.07	2.56		167.2
		CD86	3.68	1.65	2.83		109.3
	208	ISO	2.07		2.41	2.3		86.6
		CD54	3.26	1.19	2.03		185.9
		CD86	3.68	1.61	2.41		106.6
	250	ISO	2.29		2.61	2.0		100.7
		CD54	4.79	2.50	1.22		390.6*
		CD86	4.20	1.91	2.06		126.5
	300^PS	ISO	2.68		3.52	2.5		79.4
		CD54	26.61	23.93	1.72		3739.1*
		CD86	7.83	5.15	2.23		341.1*

^PSPhase separation (oily droplets)

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

Table 5. Results of the third h-CLAT run

	Concentration (µg/mL)	Antibody / ISO	MFI GeoMean(FITC)	MFI-ISO	Cyto(Geo) GeoMean(7-AAD)	Mean Cyto	RFI (%)	Viability (%)
Negative control	-	ISO	2.17		3.09	3.1		100.0
		CD54	2.99	0.82	2.90		100.0
		CD86	3.67	1.50	3.23		100.0
Vehicle control	-	ISO	2.12		3.02	2.9		100.0
		CD54	3.03	0.91	2.77		100.0
		CD86	3.35	1.23	2.79		100.0
Positive control	2	ISO	2.58		4.09	3.9		73.5
		CD54	6.47	3.89	4.16		427.5*
		CD86	6.31	3.73	3.43		303.3*
	3	ISO	2.87		4.64	3.9		72.6
		CD54	7.34	4.47	3.64		491.2*
		CD86	8.42	5.55	3.54		451.2*
Test substance	84	ISO	2.42		3.15	3.0		96.6
		CD54	3.31	0.89	2.93		97.8
		CD86	3.37	0.95	2.80		77.2
	100	ISO	2.48		3.06	2.9		97.7
		CD54	3.34	0.86	2.88		94.5
		CD86	3.62	1.14	2.84		92.7
	121	ISO	2.53		3.25	3.0		96.5
		CD54	3.52	0.99	2.81		108.8
		CD86	3.89	1.36	2.83		110.6
	145	ISO	2.69		3.40	3.2		90.0
		CD54	4.02	1.33	3.10		146.2
		CD86	4.43	1.74	3.03		141.5
	174	ISO	2.71		3.78	3.5		82.3
		CD54	4.06	1.35	3.29		148.4
		CD86	4.40	1.69	3.36		137.4
	208	ISO	2.87		3.96	3.6		78.4
		CD54	4.94	2.07	3.58		227.5*
		CD86	5.39	2.52	3.40		204.9*
	250	ISO	3.03		4.60	4.2		67.5
		CD54	5.15	2.12	4.15		233.0*
		CD86	6.49	3.46	3.97		281.3*
	300^PS	ISO	3.23		5.58	4.9		58.0
		CD54	6.02	2.79	4.72		306.6*
		CD86	8.71	5.48	4.49		445.5*

^PSPhase separation (oily droplets)

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

Phase separation (oily droplets) was observed at the highest tested test item concentration (300 µg/mL) therefore this concentration was excluded from the evaluation. After exclusion of this concentration, the RFI of CD86 and CD54 was greater than 150% and 200%, respectively in at least one dose in 2 out of 3 independent run data.

Interpretation of results:

other: activation of dendritic cells positive according to OECD 442E

Conclusions:

Under the conditions of the Human Cell Line Activation Test the test substance increased the expression of CD86 and CD54, cell surface marker associated with the process of activation of monocytes and dendritic cells. Based on this result, the h-CLAT prediction is considered positive for skin sensitisation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitizing potential of the test substance was evaluated in a combination of non-animal methods (in chemico and in vitro). Three key events of skin sensitisation a) molecular interaction with skin proteins, b) inflammatory response in keratinocytes and c) activation of dendritic cells were addressed.

a) Molecular interaction with skin proteins

The protein reactivity of the test substance was investigated in a Direct Peptide Reactivity Assay (DPRA) (2016) performed according to OECD Guideline 442C and in compliance with GLP. The test substance was dissolved in acetonitrile to prepare a 100 mM solution. The solubility in acetonitrile was confirmed. Stock solutions of cysteine and lysine containing synthetic peptides with a purity in the range of 90-95% were freshly prepared just before incubation with the test substance. The cysteine and lysine peptide solutions were incubated in HPLC vials with the test substance or the positive control cinnamic aldehyde at 1:10 (5 mM cysteine and 5 mM test substance/positive control) and 1:50 ratios (0.5 mM lysine and 25 mM test substance/positive control), respectively. After an incubation time for a minimum of 22 h at 25 °C in an HPLC autosampler, HPLC-UV analysis for the cysteine and lysine peptides were performed. All analytical acceptance criteria for each peptide run were met and the positive control proved the validity of the test. In presence of the test substance the mean depletion of cysteine and lysine peptide was 0.906% and -1.68%, respectively. The overall mean percent cysteine and lysine depletion is 0.453 and therefore the test substance is allocated to the “no or minimal reactivity” class (mean % depletion <6.38%) and considered to be negative for molecular interaction with skin proteins.

 

b) Activation of keratinocytes

The activation of keratinocytes of the test substance was investigated in an ARE-Nrf2 Luciferase Test (2016) in the transgenic KeratinoSens™ cell line according to OECD Guideline 442D and in compliance with GLP. Cells were incubated with test substance concentrations of 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 61.5, 125, 250, 500, 1000, 2000 µM in DMSO for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement. The study was conducted in two independent experiments. In the first experiment a max luciferase activity (I_max) induction of 0.98 was determined at a test substance concentration of 1000 µM. The corresponding cell viability was 105.1%. In the second experiment a max luciferase activity (I_max) induction of 1.22 was determined at test substance concentrations of 250 and 500 mM. The corresponding cell viabilities were 111.4% and 109.7%. Therefore in both experiments no luciferase activity induction >1.5 was observed and no EC_1.5 could be calculated. Cytotoxicity (cell viability <70%) was observed at 2000 µM. Under the conditions of the ARE-Nrf2 Luciferase test the test substance did not induce luciferase activity in the transgenic KeratinoSens™ cell line.

 

c) Activation of dendritic cells

Dendritic cell response of the test substance was investigated in a human Cell Line Activation Test (h-CLAT) according to OECD Guideline 442E and in compliance with GLP. The doses for the main experiment were previously determined by two XTT tests. Cytotoxic effects were not observed following incubation with the test substance up to the highest soluble concentration of 250 μg/mL and thus a CV75 value could not be calculated. The viability of the cells in both XTT spaced within a range of 92.67% to 112.4% and 81.35% to 108.61%, respectively (threshold of cytotoxicity: < 75%).

In the main experiment test substance concentrations (solved in DMSO) of 84, 100, 121, 145, 174, 208, 250 and 300 µg/mL were administered to human THP-1 cells for 24 hours. Thereafter the cells were stained with FITC-labeled anti-CD86, CD54 antibody or mouse IgG1 (isotype control) and relative fluorescence intensity of the surface markers was measured in a flow cytometer. Since phase separation (oily droplets) was observed at the highest tested test item concentration (300 μg/mL), this concentration was excluded from the evaluation. Relative fluorescence intensity of CD86 and CD54 was greater than 150% and 200%, respectively in at least one dose in 2 out of 3 independent experiments. Therefore, the h-CLAT prediction for activation of dendritic cells is considered positive.

 

Conclusion

Based on a weight of evidence approach considering one positive and two negative results in the in vitro test battery according to the Adverse Outcome Pathway defined for skin sensitisation, the test substance is not considered to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on skin sensitisation of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.