ethyl 2-[[(1R,2S,5R)-5-methyl-2-propan-2-ylcyclohexanecarbonyl]amino]acetate

Endpoint summary

Description of key 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 were evaluated: 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) (2017) performed according to OECD Guideline 442C and in compliance with GLP.

WS-5 was dissolved in acetonitrile at a concentration of 100 mM. The test solutions were incubated at 1:10 and 1:50 ratios with the cysteine and lysine peptides, respectively, for 24±2 hours

in glass autosampler vials, protected from light and set at 25°C. Relative peptide concentration was measured by high performance liquid chromatography (HPLC) with UV detection.

Cysteine and lysine peptide percent depletion (PPD) values were then calculated and used in a prediction model which allows assigning the test article to one of four reactivity classes used

to support the discrimination between sensitisers and non-sensitisers.

In presence of the test substance, WS-5, the percentage peptide depletion for cysteine was 7.16% and the percentage peptide depletion for lysine was 0.3%. The mean of cysteine and lysine percentage depletion was therefore 3.73%. All analytical acceptance criteria for each peptide run were met and the positive control proved the validity of the test.

The test article, WS-5, was therefore considered to have no or minimal reactivity and gave a negative prediction in the Direct Peptide Reactivity Assay.

 

b) Activation of keratinocytes

The activation of keratinocytes of the test substance, WS-5 was investigated in an ARE-Nrf2 Luciferase Test (2017) in the transgenic KeratinoSens™ cell line according to OECD Guideline 442D and in compliance

with GLP. For each repetition, three replicates were used for the luciferase activity measurements and one parallel replicate used for the cell viability assay.The study was conducted in two independent experiments.

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.

In the first experiment a max luciferase activity (Imax) induction of 3.48 was determined at a test substance concentration of 2000µM (EC1.5). Cytotoxicity cell viability of 33.1% was observed at 2000 µM.

No dose response for luciferase Induction was noticed.

In the second experiment, an Imax induction of 1.25 was determined at test substance concentrations of 250 µM. The corresponding cell viability was >70%.No EC1.5 could be calculated as there no statistically

significant increases in induction.

Under the conditions of the ARE-Nrf2 Luciferase test the test substance, WS-5 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 aim of the study was to investigate the potential of WS‑5 to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1, by quantifying changes

in the expression of cell surface markers (CD86 and CD54).

A reactivity check was performed two weeks after thawing using the following compounds:

-         2,4-dinitrochlorobenzene(DNCB, CAS No. 97-00-7, ≥99% purity)

-         nickel sulphate (CAS No. 10101-97-0, 99% purity)

-         lactic acid (CAS No. 50-21-5, 85% purity).

Only cells which passed the reactivity check were used for the assay.

WS-5 was dissolved in DMSO to give working solutions at concentrations of 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL. The top two concentrations following dilution in culture medium (500 and 1000 µg/mL) produced oily emulsions and were not used for cell treatment. Therefore, the maximum attainable concentration was therefore 250 µg/mL.

No CV75 value was calculated, as there was no effect of the treatment on cell viability.

In the main experiment test substance concentrations of69.77, 83.72, 100.46, 100.46, 120.56, 144.67, 173.61, 208.33 and 250.00were 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.

In the first Experiment, a stable dispersion was not formed during the dilution steps at concentrations of 208.33 and 250 µg/mL, therefore the maximum concentration treated was 173.61 µg/mL. The relative fluorescence intensity (RFI) values for the test article, WS-5 were calculated.

The RFI of CD86 was ≥150% at concentrations of 120.56, 144.67 and 173.61 µg/mL in the first Experiment, with viability ≥50%. The RFI of CD54 was≥200% at concentrations of 144.67 and 173.61 µg/mL, with viability ≥50%.

In second Experiment, the RFI of CD86 was ≥150% at concentrations of 69.77, 83.72, 100.46, 120.56, 144.67 and 173.61µg/mL, with viability≥50%. The RFI of CD54 was ≥200% at concentrations of 69.77, 83.72, 100.46, 120.56, 144.67 and 173.61µg/mL, with viability ≥50%.

The assay acceptance criteria required for acceptance of results in the test were satisfied according to OECD Test Guideline 422E.

Based on the results of this study performed according to OECD TG 442, the test article, WS‑5, was considered to be positive in the human Cell Line Activation Test.

 

 

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 sensitization, the test substance, WS-5 is not considered to be a skin sensitizer.

 

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

The OECD TG 442 C may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: white crystalline powder, WS-5, batch no.: 80100039
- Expiration date of the batch: 16 January 2019
- Purity test date: 99.57%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry ambient temperature

The test article, WS-5 produced a visually clear solution at a concentration of 100 mM acetonitrile, which was the first of the listed vehicles specified in the protocol.
Formulations were prepared shortly before testing.

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:

Test Article Incubation:
Each test solution was prepared at ratios of 1:10 and 1:50 with the cysteine and lysine stock solutions, respectively. The preparations were placed in an incubator set at 25°C for 24±2 hours, in the dark. At the end of the incubation period the samples were visually inspected for precipitate formation. Samples were centrifuged at 400 g for 5
minutes.

Analytical Method:
The remaining concentration of cysteine- or lysine-containing peptides following the 24-hour incubation period was measured by high performance liquid chromatography
(HPLC) with gradient elution and UV detection at 220 nm.

Reference and Co-elution Controls:
Reference controls were prepared for each peptide.
Reference Control A (in triplicate) was used to verify the HPLC system suitability prior to the analysis. Reference Control B (six replicates) was used to verify the
stability of the reference controls over time and Reference Control C (in triplicate) was used to verify that acetonitrile did not impact the percent peptide depletion.
Co-elution controls were prepared to detect possible co-elution of the test article with the peptides.

Calibration Curves for Peptides:
Calibration curves were prepared for each peptide using concentrations of 0.0167, 0.0334, 0.0667, 0.1335, 0.267 and 0.534 mM (Standards 1 to 6)

Positive control results:

Cinnamaldehyde (CAS No. 104-55-2, batch number MKBT8955V, purity 98.5%, expiry 29 February 2020) was used as the positive control. The positive control was dissolved in acetonitrile at a concentration of 100 mM. For results, please see table 2 and 3.

Key result

Run / experiment:

other: 3

Parameter:

other: The mean of cysteine and lysine percentage depletion

Value:

3.73

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

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

Remarks:

Negative prediction in the Direct Peptide Reactivity Assay

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The mean percent cysteine and percent lysine depletion value was calculated. Negative depletion was considered as “0” when calculating the mean. By using the
cysteine 1:10/lysine 1:50 prediction model below, the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers in the framework of an IATA.

The percentage peptide depletion for cysteine was 7.16% and the percentage peptide depletion for lysine was 0.3%. The mean of cysteine and lysine percentage depletion was therefore 3.73%.

ACCEPTANCE OF RESULTS:
The following criteria should be met for a run to be considered valid:

- The standard calibration curve should have a r2 >0.99.
The r value for the standard calibration curve was 1.000 and as 0.9963 for lysine and cyteine depletion, respectively.
- The mean peptide concentration for reference controls A should be 0.50±0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference
controls B and C should be <15.0%.
For lysine and cysteine depletion, the mean Peptide Concentration (mM) for the Reference Controls A and C were as follows:
Reference Controls A - 0.50
- The mean PPD value of the three replicates for the positive control and maximum standard deviation (SD) must fall within the ranges:

Peptide Mean PPD Values (%) Lower Bound Mean PPD Values (%) Upper Bound SD
Cysteine 60.8 100 <14.9
Lysine 40.2 69.0 <11.6

- The maximum standard deviation for the test article replicates should be <14.9 for the percent cysteine depletion and <11.6 for the percent lysine depletion.
The SD for Mean PPD for lysine and cysteine depletion were 0.81 and 2.72 , respectively.

Table 1. Cysteine 1:10/lysine 1:50 Prediction Model used in this testing to discriminate between skin sensitisers and non-sensitisers in the framework of an IATA

Mean of Cysteine and Lysine % Depletion	Reactivity Class	DPRA Prediction
0% ≤ mean % depletion ≤6.38%	No or minimal reactivity	Negative
6.38% < mean % depletion ≤22.62%	Low reactivity	Positive
22.62% < mean % depletion ≤42.47%	Moderate reactivity
42.47% < mean % depletion ≤100%	High reactivity

Table 2. The percentage peptide depletion (PPD) for lysine

Substance	Replicate Peptide Peak Areas	Reference Control C Mean Peptide Peak Area	PPD	Mean PPD	SD
WS-5 Test Article	37.49	37.26	-0.62 #	0.30	0.81
	37.41		-0.40 #
	36.93		0.89
Cinnamaldehyde Positive Control	17.63	37.26	52.68	50.35	4.61
	17.39		53.33
	20.48		45.03

#negative value was considered as “0” when calculating the mean

Table 3. The percentage peptide depletion (PPD) for cysteine

Substance	Replicate Peptide Peak Areas	Reference Control C Mean Peptide Peak Area	PPD	Mean PPD	SD
WS-5 Test Article	22.13	23.06	4.03	7.16	2.72
	20.99		8.98
	21.11		8.46
Cinnamaldehyde Positive Control	8.15	23.06	64.66	65.50	0.74
	7.89		65.78
	7.83		66.05

Interpretation of results:

other: negative prediction for skin sensitisation

Conclusions:

The percentage peptide depletion for cysteine was 7.16% and the percentage peptide depletion for lysine was 0.3%. The mean of cysteine and lysine percentage depletion was therefore 3.73%. The test article, WS-5, was therefore considered to have no or minimal reactivity and gave a negative prediction in the Direct Peptide Reactivity Assay, performed according to OECD Guidelines for Testing of Chemicals Method 442C.

Executive summary:

The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptides following incubation with the test article.

The test article, WS-5 was dissolved in acetonitrile at a concentration of 100 mM. The test solutions were incubated at 1:10 and 1:50 ratios with the cysteine and lysine peptides, respectively, for 24±2 hours in glass autosampler vials, protected from light and set at 25°C.

Relative peptide concentration was measured by high performance liquid chromatography (HPLC) with UV detection.

Cysteine and lysine peptide percent depletion (PPD) values were then calculated and used in a prediction model which allows assigning the test article to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

In presence of the test substance, WS-5, the percentage peptide depletion for cysteine was 7.16% and the percentage peptide depletion for lysine was 0.3%. The mean of cysteine and lysine percentage depletion was therefore 3.73%.

All analytical acceptance criteria for each peptide run were met and the positive control proved the validity of the test.

Conclusion:

The test article, WS-5, was considered to have no or minimal reactivity and gave a negative prediction in the Direct Peptide Reactivity Assay according to OECD TG 442 C.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 September 2017 - 22 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see any other information on materials and methods including tables

GLP compliance:

yes

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Symrise
- batch No.of test material: 80100039
- Expiration date of the lot/batch: 16 January 2019
- Purity test date: 99.57%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Analysis for stability, achieved concentration and homogeneity of test article formulations was not conducted as part of this study as it was not a requirement of the Test Guideline.
- Storage: 15 to 25°C, protected from light

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid, stock liquid or gel: WS-5 was dissolved in dimethyl sulfoxide (DMSO) to the final concentration of 200 mM. Serial dilutions were then made using DMSO to obtain 12 master concentrations of the test article (0.098, 0.196, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50, 100 and 200 mM).

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

The ARE-Nrf2 luciferase test method utilises an immortalised adherent cell line derived from HaCaT human keratinocytes. The cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 promoter fused with the ARE from a gene known to be up-regulated by contact sensitisers.

Aliquots of 50 µL of each of the final concentrations of WS-5 were transferred to each of three luciferase plates and a single viability plate. Each plate was sealed using a plate sealer and then incubated at 37±1C, 5% (v/v) CO2 in air, in a humidified environment for 48±1 hours.

After the 48-hour exposure period, the medium in the luciferase plates was replaced with fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The plate was sealed and incubated for 4 hours at 37±1°C, 5% (v/v) CO2.

The MTT medium was then removed and SDS (at 10% w/v) was added per well. The plate was sealed and placed into an incubator at 37±1°C, 5% (v/v) CO2 in air and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm using a SpectraMax M2e (Molecular Devices, LLC).

After the 48-hour exposure period, the cells in the viability plate were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well.

The plates were then incubated for 20 minutes at 25±2°C, loaded into the luminescence plate reader and read.

The negative control was diluted into culture medium containing serum so that the final concentration was 1%.

The positive control was prepared at a concentration of 6.4 mM in DMSO. Five master concentrations ranging from 0.4 to 6.4 mM were prepared in DMSO (from a 6.4 mM stock solution). The master concentrations were then further diluted 25 fold into culture medium containing serum and finally used for treatment with a further 4 fold dilution factor so that the final concentrations range from 4 to 64 µM.

Positive control results:

Cinnamic aldehyde (CAS No. 14371-10-9), supplied by Sigma Aldrich Chemical Co. Ltd. was used as the positive control.

Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 16 to 64 µM in Experiments 1 and 2. The EC1.5 values for the positive control were 8.26 and 11.54 µM in Experiments 1 and 2, respectively. The average induction in the two replicates for the positive control at 64 µM were 10.96 and 3.81 in Experiments 1 and 2, respectively.

Key result

Run / experiment:

other: 3 runs / Experiment 1

Parameter:

other: maximum luciferase activity induction (I max)

Value:

3.48

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: negative in the KeratinoSens™ prediction model

Remarks:

Corresponding cytotoxicity cell viability of 33.1%. EC1.5 at 2000 µM. No dose response for luciferase Induction

Key result

Run / experiment:

other: 3 runs / Experiment 2

Parameter:

other: maximum luciferase activity induction (I max)

Value:

1.25

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: negative in the KeratinoSens™ prediction model

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prediction model:
A KeratinoSens™ prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSens™ prediction is considered negative:
1.The Imax is >1.5 fold and statistically significantly different when compared to the solvent (negative) control (se determined by a two-tailed, unpaired Student’s T-test);
2.The cell viability is >70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration);
3.The EC1.5 value is <1000 µM (or <200 µg/mL for test articles with no defined MW);
4.There is an apparent overall dose response for luciferase induction or if the dose response curve is biphasic.

   

Table1. Luminescence Readingsfor WS-5 - Experiment 1
Substance		Concentration (µM)
		0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000 #	2000
Test Article	Plate 1	1133240	843755	898692	1050636	786297	718257	847774	1019132	995454	1002721	0	2538667
	Plate 2	957910	983945	957037	746169	898212	938561	995668	1078826	1324627	1037682	0	2944623
	Plate 3	799640	871633	753495	860062	719102	716173	809385	898619	890782	1247988	0	3350900
	Mean Fold Induction	1.14	1.06	1.02	1.06	0.94	0.93	1.04	1.18	1.25	1.30	0.00	3.48

# no luminescence readings obtained due to the technical error

 

Table 2. Luminescence Readings forNegative Control- Experiment 1
Substance		Individual Values
Negative Control	Plate 1	744394	817814	829305	811138	840569	737785
	Plate 2	784779	1045144	1048312	887335	958900	1010644
	Plate 3	738233	775860	880078	798360	928753	684907

 

Table 3. Luminescence Readings forPositive Control- Experiment 1
Substance		Concentration (µM)
		4	8	16	32	64
Positive Control	Plate 1	1209862	1164304	1988218	3053730	6682098
	Plate 2	1155343	1500502	1891800	2592479	12724356
	Plate 3	1043222	1127017	1493561	2641184	8953845
	Mean Fold Induction	1.34	1.48	2.11	3.28	10.96

 

 

Table 4. Luminescence Readings for WS-5 - Experiment 2
Substance		Concentration (µM)
		0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Test Article	Plate 1	1152178	975059	926744	883957	884997	866903	906172	999005	1044621	1086959	17853	-297
	Plate 2	1041039	921073	936655	915797	870204	900632	853558	981584	1048706	1174808	21617	-2
	Plate 3	963458	842209	891649	885147	879821	811156	884135	962980	983201	1083250	908870	68
	Mean Fold Induction	1.18	1.02	1.03	1.01	0.99	0.96	0.99	1.10	1.15	1.25	0.37	0.00

 

Table 5. Luminescence Readings for Negative Control - Experiment 2
Substance		Individual Values
Negative Control	Plate 1	980930	932116	843684	915855	910491	945191
	Plate 2	974030	847451	858485	887633	845526	924225
	Plate 3	895280	843147	760858	867474	912730	890969

 

Table 6. Luminescence Readings for Positive Control - Experiment 2
Substance		Concentration (µM)
		4	8	16	32	64
Positive Control	Plate 1	1053652	1258489	1341586	2106567	3757925
	Plate 2	1038281	1213899	1523531	1989432	3401688
	Plate 3	1078725	1320105	1405587	2034596	3037362
	Mean Fold Induction	1.19	1.42	1.60	2.29	3.81

 

 

 

Interpretation of results:

other: negative prediction using the KeratinoSens™ prediction model

Conclusions:

Under the conditions of the ARE-Nrf2 Luciferase test, the test substance, WS-5 did not induce luciferase activity in the transgenic KeratinoSens™ cell line.

Executive summary:

This study according to OECD TG 442D was conducted to investigate the potential ofWS-5to induce genes that are regulated by the antioxidant response element (ARE).

The applied ARE-Nrf2 Luciferase Test method utilises an immortalised adherent cell line derived from HaCaT human keratinocytes. The cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 promoter fused with the ARE from a gene known to be up-regulated by contact sensitisers.

The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic substances.

For each repetition, three replicates were used for the luciferase activity measurements and one parallel replicate used for the cell viability assay.The study was conducted in two independent experiments.

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.

In the first experiment a max luciferase activity (Imax) induction of 3.48 was determined at a test substance concentration of 2000µM (EC_1.5). Cytotoxicity cell viability of 33.1% was observed at 2000 µM. No dose response for luciferase Induction was noticed.

In the second experiment, an Imax induction of 1.25 was determined at test substance concentrations of 250 µM. The corresponding cell viability was >70%.No EC1.5 could be calculated as there no statistically significant increases in induction.

Conculsions:

Under the conditions of the ARE-Nrf2 Luciferase test the test substance, WS-5 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:

key study

Study period:

09 October 2017 - 27 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals Method 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))

Deviations:

not specified

GLP compliance:

yes

Type of study:

other: human Cell Line Activation Test (h - CLAT)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Symrise
- batch No.of test material: 80100039
- Expiration date of the lot/batch: 16 January 2019
- Purity test date: 99.57%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Analysis for stability, achieved concentration and homogeneity of test article formulations was not conducted as part of this study as it was not a requirement of the Test Guideline.
- Storage: 15 to 25°C, protected from light

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid, stock liquid or gel: the test article, WS-5, was dissolved at 500 mg/mL in DMSO. Eight stock solutions were prepared by 2-fold serial dilutions using the corresponding solvent.
The stock solutions were further diluted 250-fold in culture medium to give working solutions at concentrations of 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL.
The top two concentrations following dilution in culture medium (500 and 1000 µg/mL) produced oily emulsions and were not used for cell treatment. Therefore, the maximum attainable concentration was therefore 250 µg/mL.

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

In a performed Dose Finding Assay, an oily emulsion was observed during the dilution steps at concentrations of 500 and 1000 µg/mL, therefore the maximum attainable concentration was 250 µg/mL. No CV75 value was calculated, as there was no effect of the treatment on cell viability.

CD86/CD54 Expression procedure:
Eight stock solutions of WS-5 were prepared by 1.2-fold serial dilutions using DMSO to give eight concentrations ranging from 34.9 to 125 mg/mL. These stock solutions were then diluted 250-fold into the culture medium (working solutions).

Aliquots of 500 µL of each of the working solutions were mixed 1:1 with cell suspensions at 1 x 106 cells per well. Each plate was sealed using a plate sealer and then incubated at 37±1C, 5% (v/v) CO2 in air, in a humidified environment for 24±0.5 hours.

After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation, washed with FACS buffer and then blocked with 600 µL of blocking solution (FACS buffer containing 0.01% (w/v) globulin) at 4C for 15 minutes.

After blocking, the cells were split into three aliquots of 180 µL into a 96-well plate (or sample tubes), centrifuged and then stained with FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies at 4C for 30 minutes.

The stained cells were washed with FACS buffer and re-suspended in FACS buffer and propidium iodide solution was added. The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

Positive control results:

For the positive control, 2,4-dinitrochlorobenzene (DNCB, CAS No. 97-00-7, ≥99% purity), RFI values were ≥150% for CD86 and ≥200% for CD54 in all each independent runs. Cell viability was >50% in each independent run.

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: relative fluorescence intensity (RFI)

Remarks:

RFI of CD86

Value:

150

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: activation of dendritic cells positive according to OECD 442E at concentrations of 120.56, 144.67 and 173.61 µg/mL

Remarks:

viability ≥50%.

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: relative fluorescence intensity (RFI)

Remarks:

RFI of CD86

Value:

150

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: activation of dendritic cells positive according to OECD 442E at concentrations of 69.77, 83.72, 100.46, 120.56, 144.67 and 173.61 µg/mL

Remarks:

viability ≥50%

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: relative fluorescence intensity (RFI)

Remarks:

RFI of CD54

Value:

200

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: activation of dendritic cells positive according to OECD 442E at concentrations of 144.67 and 173.61 µg/mL

Remarks:

viability ≥50%

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: relative fluorescence intensity (RFI) RFI of CD54

Remarks:

RFI of CD54

Value:

200

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: activation of dendritic cells positive according to OECD 442E at concentrations of 69.77, 83.72, 100.46, 120.56, 144.67 and 173.61 µg/mL

Remarks:

viability ≥50%

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Assay Acceptance Criteria
- The cell viabilities of medium and solvent control should be higher than 90%.
- In the solvent control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%).
- For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be >105%.
- 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%.
- For the test article, the cell viability should be more than 50% in at least four tested doses in each run.

ACCEPTANCE OF RESULTS:
- The cell viabilities of medium and solvent control were higher than 90% in each independent run.
- In the solvent control, RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%). The assay was therefore considered valid as this did not impact on the results obtained with the test article, WS-5.
- For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control was >105% on all occasions.
- For the positive control, RFI values were ≥150% for CD86 and ≥200% for CD54 in all each independent runs. Cell viability was >50% in each independent run.
- For the test article, WS-5, the cell viability was more than 50% in all tested concentrations in each independent run.

Table 1. Dose Finding Assay: Cell Viability
Run	Cell Viability (%) at Concentration (µg/mL)
	7.8	15.6	31.3	62.5	125.0	250.0
1	99.7	99.7	99.5	99.2	96.6	95.2
2	99.6	99.4	99.2	98.6	96.3	96.7

Table 2. Experiment 1 - MFI and Cell Viability Values
WS-5 Concentration (µg/mL)	MFI (Geo Mean)			Corrected MFI		Cell Viability
	CD86	CD54	Isotype	CD86	CD54	IgG	CD86	CD54
69.77	1677	878	608	1069	270	97.0	96.5	97.2
83.72	1714	866	628	1086	238	96.9	96.1	96.8
100.46	1844	917	629	1215	288	95.4	94.8	94.8
120.56	2100	955	629	1471	326	90.9	89.6	91.0
144.67	2142	982	635	1507	347	91.0	89.4	87.5
173.61	2273	1117	652	1621	465	83.0	82.6	80.5
Medium	1352	731	594	758	137	98.8	98.6	98.9
DMSO	1486	750	586	900	164	98.8	98.4	98.7
DNCB	2291	1269	759	1532	510	86.5	86.8	88.8

MFI (Geo Mean) Geometric mean fluorescence intensity

Corrected MFI Corrected mean fluorescence intensity

Table 3. Experiment 2 -MFI and Cell Viability Values
WS-5 Concentration (µg/mL)	MFI (Geo Mean)			Corrected MFI			Viability
	CD86	CD54	Isotype		CD86	CD54	Isotype	CD86	CD54
69.77	1848	906	637		1211	269	93.8	91.9	91.6
83.72	1959	1003	649		1310	354	91.9	88.8	88.9
100.46	2097	1020	658		1439	362	90.7	88.3	87.5
120.56	2336	1136	684		1652	452	83.9	77.8	77.9
144.67	2240	1176	708		1532	468	78.0	74.0	72.8
173.61	2278	1259	741		1537	518	70.0	63.5	62.1
208.33	2210	1256	836		1374	420	40.9	40.7	41.3
250.00	2620	1261	919		1701	342	24.6	21.8	24.0
Medium	1086	694	578		508	116	99.2	99.1	98.7
DMSO	1102	690	571		531	119	99.0	98.8	98.8
DNCB	2281	1341	750		1531	591	84.6	82.2	79.7

MFI (Geo Mean)                Geometric mean fluorescence intensity

Corrected MFI                    Corrected mean fluorescence intensity

 

Table 4. The relative fluorescence intensity (RFI) values for WS-5
Concentration (µg/mL)	RFI (CD86)		RFI (CD54)
	Exp 1	Exp 2	Exp 1	Exp 2
69.77	119	228	165	226
83.72	121	247	145	297
100.46	135	271	176	304
120.56	163	311	199	380
144.67	167	289	212	393
173.61	180	289	284	435
208.33	-	259	-	353
250.00	-	320	-	287

Interpretation of results:

other: positive prediction of skin sensitisation in the human Cell Line Activation Test.

Conclusions:

Based on the results of this study performed according to OECD TG 442, the test article,WS-5, was considered to be positive in the human Cell Line Activation Test.

Executive summary:

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 aim of the study was to investigate the potential of WS‑5 to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54).

A reactivity check was performed two weeks after thawing using the following compounds:

-    2,4-dinitrochlorobenzene (DNCB, CAS No. 97-00-7, ≥99% purity)

-    nickel sulphate (CAS No. 10101-97-0, 99% purity)

-    lactic acid (CAS No. 50-21-5, 85% purity).

Only cells which passed the reactivity check were used for the assay.

WS-5 was dissolved in DMSO to give working solutions at concentrations of 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL. The top two concentrations following dilution in culture medium (500 and 1000 µg/mL) produced oily emulsions and were not used for cell treatment. Therefore, the maximum attainable concentration was therefore 250 µg/mL.No CV75 value was calculated, as there was no effect of the treatment on cell viability.

In the main experiment, the test substance concentrations of 69.77, 83.72, 100.46, 100.46, 120.56, 144.67, 173.61, 208.33 and 250.00 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.

In the first Experiment, a stable dispersion was not formed during the dilution steps at concentrations of 208.33 and 250 µg/mL, therefore the maximum concentration treated was 173.61 µg/mL. The relative fluorescence intensity (RFI) values for the test article, WS-5 were calculated.

The RFI of CD86 was ≥150% at concentrations of 120.56, 144.67 and 173.61 µg/mL in the first Experiment, with viability ≥50%. The RFI of CD54 was≥200% at concentrations of 144.67 and 173.61 µg/mL, with viability ≥50%.

In second experiment, the RFI of CD86 was ≥150% at concentrations of 69.77, 83.72, 100.46, 120.56, 144.67 and 173.61µg/mL, with viability ≥50%. The RFI of CD54 was ≥200% at concentrations of 69.77, 83.72, 100.46, 120.56, 144.67 and 173.61µg/mL, with viability ≥50%.

The assay acceptance criteria required for acceptance of results in the test were satisfied according to the OECD Test Guideline 422E.

Conclusions:

Based on the results of this study performed according to OECD TG 442, the test article, WS‑5, was considered to be positive in the human Cell Line Activation Test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin sensitisation results:

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

Skin sensitisation (OECD 442E): 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, WS-5 is not considered to be a skin sensitiser.

The available data on skin sensitisation of the test substance do not meet the criteria for classification according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.