Alcohols, C12-14 (even numbered), propoxylated, aminated, ethoxylated

Endpoint summary

Description of key information

Two in vitro assays (OECD 442 D and OECD 442 E ) and one in chemico assay (OECD 442C) were performed with the test item.

The DPRA (OECD 442C) and the KeratinoSens (OECD 442D) were negative for skin sensitization. The result of the hCLAT assay (OECD 442E) was positive. However, the test item is a skin irritant, has limited solubility and it is relatively cytotoxic which might impact the outcome of the hCLAT assay.  The information from the three in vitro/ in chemico assays was integrated using the defined approach "2 out of 3" published in the Draft OECD Guideline Defined Approaches for Skin Sensitisation (OECD, December 2020). This defined appraoch (DA) integrates the outcome of the assays and predicts if a substnace is a skin sensitiser or non-sensitiser. The DA's balanced accuracy is 83.5% to predict the LLNA and 88.4% to predict sensitisation in human. The prediction for our test item is non sensitiser. The conclusion is that the test material is a non-sensitiser and the data is not sufficient for classification.

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:

key study

Study period:

Experimental start date: 26 November 2018 and Experimental completion date: 13 December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

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

Details on the study design:

Cell Culture:
The cells used in this assay were the transgenic cell line KeratinoSens™ with a stable insertion of the luciferase construct supplied by Givaudan (Dubendorf, Switzerland). The cells were routinely grown and subcultured in maintenance medium at 37°C ± 2°C in a humidified atmosphere containing 5% CO2 in air.

Cell Culture from Frozen Stocks
Vials of KeratinoSens™ cells, stored frozen in cryotubes at -196°C under liquid nitrogen

Cell Passage
Actively growing cell stocks were maintained and expanded by subculturing (passage). When the cells had reached 80 – 90% confluence, the medium from each flask was removed and harvested using trypsin-EDTA solution. Cultures were incubated at 37 ± 2°C in a humidified atmosphere containing 5% CO2 in air until complete detachment and disaggregation of the cell monolayer had occurred. The cells were then resuspended in medium to neutralise the trypsin (cells from several flasks may have been pooled at this point). The cells were resuspended and distributed into flasks containing fresh maintenance medium. This passage procedure was repeated to provide a sufficient number of cells for a test, and were passaged at least twice before using the cells in a test. The passages of KeratinoSens™ cells were limited to 25 passages.

Preparation of Test Cell Cultures
The cells from flasks of actively growing cultures were detached and disaggregated as described above. The number of viable cells in the prepared cell suspension were determined by counting a trypan blue-stained cell preparation using an Improved Neubauer Haemocytometer. The cell suspension was diluted with maintenance medium without geneticin to give 1 x 105 viable cells/mL and 100 µL volumes pipetted into all wells except well H12 of sterile 96-well flat-bottomed microtitre plates. On each occasion four plates were prepared in parallel: three white plates for measuring luminescence and one transparent plate for measuring cell viability using the MTT assay. Well H12 of each plate received 100 µL maintenance medium without geneticin with no cells. The plates were incubated for 24 ± 2 hours at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air, to allow the cells to attach.

Preparation of the Positive Control
Cinnamic aldehyde (Sigma, 239968, lot: STBG0250V, expiry: July 2020) was prepared by weighing between 20 – 40 mg into a tared glass container and diluted to a final concentration of 200 mM in DMSO using the following formula:

V=5×((p÷100)×w) / MW- (w/1000)
Where
V = volume of DMSO in mL to be added
p = purity of the chemical in %
MW = molecular weight of the chemical in g/mol
w = exact weight of the chemical added to the vial in mg
The 200 mM cinnamic aldehyde solution was further diluted to a final concentration of 6.4 mM by adding 32 µL of the 200 mM solution to 968 µL of DMSO.

Test Item Solubility
The test item was found to be soluble in DMSO at 200 mM.

Preparation of the Test Item
A stock solution of the test item was prepared by weighing between 20 – 40 mg into a tared glass container and diluting to 200 mM in DMSO using the formula above.

V=5×(((p÷100)×w) / MW)- (w/1000)

Where
V = volume of DMSO in mL to be added
p = purity of the chemical in %
MW = molecular weight of the chemical in g/mol
w = exact weight of the chemical added to the vial in mg
The 200 mM cinnamic aldehyde solution was further diluted to a final concentration of 6.4 mM by adding 32 µL of the 200 mM solution to 968 µL of DMSO.


Preparation of the 100x Solvent Plate
A 100x solvent plate was set up by adding 100 µL of DMSO to all wells of a 96 well plate except wells in column 12 and well H11 of the plate in test 1 and well B11 of the plate in test 2. 200 µL of the stock solution of the test item, Alcohols, C12-14 alkyl ethers, propoxylated, aminated, ethoxylated (XTJ-785, Experimental), was added to one well in column 12. The test item was serially diluted across the plate by transferring 100 µL from column 12 to column 11 and then mixed by repeat pipetting (at least 3 times) and then 100 µL was transferred from column 11 to column 10 and so forth across the plate.
200 µL of the 6.4 mM stock solution of cinnamic aldehyde was added to well H11 in test 1 and well B11 of the plate in test 2 and serially diluted from column 11 to column 7.


Preparation of the Dilution Plate
The 100x solvent plate was replicated into a fresh 96 well plate by adding 240 µL of assay medium to each well and then 10 µL solution per well from the 100x solvent plate was added to equivalent wells on the dilution plate. Assay medium was 495 mL DMEM (Gibco 21885), supplemented with 5.0 mL FBS.


Treatment of Cultured Plates
Approximately 24 hours after the test cell culture plates were established, the medium was removed from the wells by careful inversion of the plates and blotting onto sterile paper towel. 150 µL of assay medium was added to every well of the 96 well plates. 50 µL from each well of the dilution plate was transferred to equivalent wells in the 96 well plates. Three white plates were dosed for measuring luminescence and one transparent plate for measuring cell viability using the MTT assay.
The plates were then covered with a plate seal and placed in the incubator at 37 ± 2°C, in a humidified atmosphere of 5% CO2 in air for 48 ± 2 hours.


Cell Viability Measurement
After incubation, the transparent plate was removed from the incubator and the plate seal discarded. The cell culture medium was removed by careful inversion of the plate and blotted onto sterile paper towel to remove residual culture medium. 100 µL fresh assay medium was added to each well. 10 µL of MTT solution (5 mg/mL in PBS) was added to each well of the 96-well plate. The plate was incubated at 37 ± 2C in a humidified atmosphere of 5% CO2 in air for 4 hours ± 10 minutes. The medium was then removed by careful inversion of the plate and blotted onto sterile paper towel to remove residual culture medium. 50 µL of DMSO was added to each well. The plate was then placed in the incubator at 37 ± 2°C, in a humidified atmosphere of 5% CO2 in air, protected from light, for at least 10 minutes. The absorbance value of each well was read using a plate reader with a 540 nm filter.


Luciferase Measurement
Luciferase was measured using the Steady Glo® Luciferase Assay system kit supplied by Promega (E2550). Steady-Glo® luciferase reagent was prepared by transferring the contents of one bottle of Steady-Glo® buffer to one bottle of Steady-Glo® substrate. The reagent was mixed by inversion until the substrate had dissolved. The reconstituted reagent was used on the same day it was prepared for the repeat of test 1 and for test 2. Frozen reconstituted reagent was used for test 1 and was thawed to room temperature before use.
After incubation the medium was removed from the wells of the triplicate white plates by careful inversion of the plates and blotting on sterile absorbent paper. 100 µL of fresh assay medium was added to each well before 100 µL of Steady-Glo® luciferase reagent was added to each well of the plate. The plates were shaken on a plate shaker for at least 5 minutes until the cells had lysed. Luminescence (emitted light) was measured using a SpectraMax L luminometer. Each plate was read for total photon count with an integration time of 1 second. The plates were dark adapted for 1 minute prior to measurement.

Number of Tests Required
Two independent tests each containing three replicates (i.e. n=6) were required to make a conclusion.

Repeat Test
The first test was conducted between 26 November 2018 and 29 November 2018, however, the results for the average coefficient of variation of the luminescence reading for the DMSO solvent control was above than the acceptance criterion of below 20%. The first test was repeated between 03 December 2018 and 06 December 2018. Only the data and results from the repeat test are included in this report.

Positive control results:

The luciferase activity induction obtained with the positive control, cinnamic aldehyde, was statistically significant above the threshold of 1.5 in at least one of the tested concentrations (4 to 64 μM) in both tests.
The EC1.5 values of the positive control, cinnamic aldehyde were 11.24 μM and 10.84 μM for test 1 and 2, respectively, which lay within the historical control range for this laboratory (see Table 5). The average induction in the three replicates for cinnamic aldehyde at 64 µM were 5.09 and 4.15 for test 1 and 2, respectively, which met the acceptance criterion of between 2 and 8.

Key result

Run / experiment:

other: test 1

Parameter:

other: I max for test item

Value:

0.69

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: The Imax tests was <1.5 fold compared to the DMSO control and therefore the EC1.5 could not be calculated.

Key result

Run / experiment:

other: test 2

Parameter:

other: I max for test item

Value:

0.94

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: The Imax tests was <1.5 fold compared to the DMSO control and therefore the EC1.5 could not be calculated.

Key result

Run / experiment:

other: test 1

Parameter:

other: cellular viability in %

Value:

70

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: The lowest two concentrations were non-toxic.

Key result

Run / experiment:

other: test 2

Parameter:

other: cellular viability in %

Value:

70

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: The lowest two concentrations were non-toxic.

Key result

Run / experiment:

other: test 1 and test 2

Parameter:

other: IC30

Value:

2.49

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: µM

Key result

Run / experiment:

other: test 1

Parameter:

other: IC50

Value:

2.89

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: µM

Key result

Run / experiment:

other: Test 2

Parameter:

other: IC50

Value:

2.92

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: µM

Other effects / acceptance of results:

Test item results:
The Imax for the test item was 0.69 in test 1 and 0.94 in test 2.
The Imax tests was <1.5 fold compared to the DMSO control and therefore the EC1.5 could not be calculated.
he cellular viability at the highest ten concentrations fell below 70% in both tests. The lowest two concentrations were non-toxic.
The IC30 value was 2.49 µM in both tests and the IC50 values were 2.89 µM and 2.92 µM in tests 1 and 2, respectively.


Negative solvent results.
The average coefficient of variation of the luminescence reading for the negative solvent control (DMSO) was 18.3% and 13.7% for test 1 and 2, respectively, which met the acceptance criterion of below 20%.

Results for test item – Test 1

Test item conc. (µM)	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Mean fold induction	0.45	0.69	0.70	0.04	-0.01	0.01	-0.01	0.01	0.00	0.00	0.04	0.02
Statistically significant	No	No	No	No	No	No	No	No	No	No	No	No
Viability (%)	97.93	96.65	-0.64	-0.38	-0.70	-1.28	-1.28	-1.47	-1.02	-0.83	-1.09	-1.47
Imax	0.69*
EC1.5(µM)	N/A
IC30(µM)	2.49
IC50(µM)	2.89

 

Determination criteria for the skin sensitisation potential of the test item	Result
Is the Imax>1.5 fold and statistically significant	No
Is the cellular viability >70% at the lowest concentration at the EC1.5determining concentration	N/A
Is the EC1.5value <1000µM	N/A
Is there an apparent overall dose-response for luciferase induction	No
KeratinoSens™ prediction	Negative

 * As the I_maxvalue of 0.70 occurred at a cytotoxic concentration, it was not considered valid. The I_maxvalue was corrected to 0.69 as this was the highest induction value that occurred at a non-cytotoxic concentration.

 

 

Results for Cinnamic Aldehyde – Test 1

Positive control conc. (µM)	4	8	16	32	64
Mean fold induction	1.17	1.35	1.72	2.71	5.09
Statistically significant	No	No	Yes	Yes	Yes
Viability (%)	110.99	118.62	114.26	103.18	110.35
Imax	5.09
EC1.5(µM)	11.24
IC30(µM)	N/A
IC50(µM)	N/A

 

Test Acceptance Criteria	Result
Luciferase activity induction obtained with the positive control statistically significant above the threshold of 1.5 in at least one of the test concentrations	Yes	Pass
Average induction of positive control at 64 µM between 2 – 8	Yes (5.09)	Pass
EC1.5of positive control within two standard deviations of the historical mean (-6.79 to 42.08)	Yes (11.24)	Pass
CV% of blank values < 20%	Yes (18.3%)	Pass

 

 

Results for test item – Test 2

Test item conc. (µM)	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Mean fold induction	0.94	0.88	0.34	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
Statistically significant	No	No	No	No	No	No	No	No	No	No	No	No
Viability (%)	105.06	95.62	3.48	3.71	5.62	1.80	1.24	1.46	2.25	1.80	3.93	3.15
Imax	0.94
EC1.5(µM)	N/A
IC30(µM)	2.49
IC50(µM)	2.92

 

Determination criteria for the skin sensitisation potential of the test item	Result
Is the Imax>1.5 fold and statistically significant	No
Is the cellular viability >70% at the lowest concentration at the EC1.5determining concentration	N/A
Is the EC1.5value <1000µM	N/A
Is there an apparent overall dose-response for luciferase induction	No
KeratinoSens™ prediction	Negative

 

 

Results for Cinnamic Aldehyde – Test 2

Positive control conc. (µM)	4	8	16	32	64
Mean fold induction	1.18	1.43	1.63	2.29	4.15
Statistically significant	No	No	Yes	Yes	Yes
Viability (%)	106.07	110.00	112.70	119.21	118.09
Imax	4.15
EC1.5(µM)	10.84
IC30(µM)	N/A
IC50(µM)	N/A

 

Test Acceptance Criteria	Result
Luciferase activity induction obtained with the positive control statistically significant above the threshold of 1.5 in at least one of the test concentrations	Yes	Pass
Average induction of positive control at 64 µM between 2 – 8	Yes (4.15)	Pass
EC1.5of positive control within two standard deviations of the historical mean (-6.79 – 42.08)	Yes (10.84)	Pass
CV% of blank values < 20%	Yes (13.7%)	Pass

Historical Control Data for Cinnamic Aldehyde

n	Date	EC1.5(µM)
1	21-Jul-17	20.43
2	11-Aug-17	34.51
3	16-Nov-17	33.93
4	16-Nov-17	29.04
5	15-Dec-17	45.81
6	15-Dec-17	43.48
7	15-Feb-18	15.84
8	15-Feb-18	19.90
9	22-Feb-18	21.07
10	22-Feb-18	27.98
11	12-Jul-18	10.18
12	26-Jul-18	16.09
13	02-Aug-18	8.88
14	09-Aug-18	17.01
15	21-Sep-18	6.36
16	25-Oct-18	4.85
17	25-Oct-18	6.10
18	25-Oct-18	5.28
19	01-Nov-18	4.33
20	01-Nov-18	12.43
21	01-Nov-18	7.50
22	08-Nov-18	10.45
23	08-Nov-18	10.93
24	08-Nov-18	11.12
	Mean	17.65
	SD	12.22
Laboratory Historical Data Range (Mean +/- 2xSD)
-6.79	to	42.08

Interpretation of results:

other: Test item is negative in the ARE-Nrf2 Luciferase Test (KeratinoSens™)

Conclusions:

It was concluded that the test item gave a negative response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the tes item is not a skin sensitizer.

Executive summary:

The purpose of this study was to support a predictive, adverse-outcome-pathway based evaluation of whether the test item is likely to be a skin sensitizer using the ARE-Nrf2 Luciferase Test (KeratinoSens™).

The I_maxfor the test item was 0.69 in test 1 and 0.94 in test 2. The I_maxfor both tests was <1.5 fold compared to the DMSO control and therefore the EC_1.5could not be calculated. The cellular viability at the highest ten concentrations fell below 70% in both tests. The lowest two concentrations were non-toxic. The IC₃₀value was 2.49 µM in both tests and the IC₅₀values were 2.89 µM and 2.92 µM in tests 1 and 2, respectively. Graphs for the test item showed no overall dose-response for luciferase induction. 

All acceptance criteria for the positive control,cinnamic aldehyde,were met.

It was concluded that the test item gave a negative response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the test item is not a skin sensitizer.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 13 November 2018 and Experimental completion date: 17 November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 sensitizers and non-sensitizers for the purpose of hazard classification and labelling.

Details on the study design:

Test system:
- Synthetic peptide containing Cysteine: Ac-RFAACAA-OH (MW 752 gr/mol); supplier AnaSpec and lot 1658140.
- Synthetic peptide containing Lysine: Ac-RFAAKAA-OH (MW 777 gr/mol); supplier AnaSpec. and lot 1658141.
- Positive control: Cinnamic Aldehyde (Positive control), MW 132 gr/mol; Batch N°MKCB9907

Assessment of Test Item Solubility
The solubility of test item was assessed in acetonitrile at a nominal concentration of 100 mM..


Preparation of peptides stock solution:
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).

Preparation of Peptide Calibration Standards
Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was also prepared.

Preparation of Stability Controls and Precision Control
Stability controls (Reference Control B), precision controls of both peptides were prepared at a concentration of 0.5 mM in acetonitrile/buffer.

Preparation of Positive Control Solution and Test Item Stock Solution
The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile. A 100 mM stock solution of test item was also prepared in acetonitrile.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls
Triplicate solutions each of the positive control and test item stock solutions were diluted with the Cysteine peptide stock solution so as to prepare solutions containing 0.5 mM Cysteine and 5 mM of Cinnamic Aldehyde or 5 mM test iteml. For the co-elution control, buffer solution was used in place of the Cysteine stock solution.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co elution Controls
Triplicate solutions each of the positive control and the test item stock solution were diluted with the Lysine peptide stock solution so as to prepare solutions containing 0.5 mM Lysine and 25 mM of Cinnamic Aldehyde or 25mM of test item. For the co-elution control, buffer solution was used in place of the Lysine stock solution.

Incubation
The appearance of test iteml and positive control samples in the HPLC vials was documented following preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.

Analysis
The concentration of both the Cysteine and Lysine peptides in the presence of test item and the associated positive controls was quantified by HPLC using UV detection as detailed in the chromatographic section.

Calculations
The peak area response for the peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration. The area responses of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured. Peptide depletion was determined using the following equation:

% Peptide depletion = 100 - (Peptide peak area in replicate depletion samples (x 100) / Mean Peptide peak area of reference control samples B)

Positive control results:

see table below

Key result

Run / experiment:

other: achieved results

Parameter:

other: Mean Cysteine depletion in the presence of the test item

Value:

-1.95

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

72.9% (positive comtrol depletion)

Remarks on result:

other: %

Key result

Run / experiment:

other: achieved results

Parameter:

other: Mean Lysine depletion in the presence of the test item

Value:

-2.33

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

58.3% (positive control depletion)

Remarks on result:

other: %

Solubility Assessment

Solubility of the test item was achieved at a nominal concentration of 100 mM in acetonitrile.

Reactivity Assessment

	Peptide	Standard Linearity	Positive control depletion (%)	Reference controls	Test item
Acceptance criteria	Cysteine	r^2>0.99	60.8-100 (SD <14.9%)	0.45-0.55 mM (CV <15%)	SD <14.9%
	Lysine	r^2>0.99	40.2-69.0 (SD <11.6%)	0.45-0.55 mM (CV <15%)	SD <11.6%
Achieved results	Cysteine	r^2>0.999	72.9 (SD, 0.06%, n=3)	B: 0.510 mM (CV 0.36%, n=6)	SD 0.13% (n=3)
	Lysine	r^2>0.999	58.3 (SD, 0.26%, n=3)	B: 0.503 mM (CV 0.44%, n=6)	SD 0.16% (n=3)

CV: Coefficient of Variation

SD: Standard deviation

The depletion of peptide in the presence of the test item was:

		Mean peak area of peptide with test item(µV.sec)	Mean peptide depletion by Test Item (%)
Cysteine	Control B: 806230 (n=6)	821960 (n=3)	-1.95
Lysine	Control B: 778780 (n=6)	796890 (n=3)	-2.33

Applying the following reactivity prediction depletion model (below), reactivity of test item is classed as “no to minimal” and the DPRA prediction is therefore negative and is therefore predicted not to be a potential skin sensitizer. 

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

There was no co-elution peaks in either the Cysteine or Lysine assay.   

Historic Control Data

Table A: Historic Data for Reference Controls

	Cysteine peptide concentration (µg/mL)^1	Lysine peptide concentration (µg/mL)^2
Maximum	386	400
Minimum	364	382
Mean	378	393
Number	36	36
CV (%)	1.27	1.24

¹                          Samples prepared at a concentration of 376 µg/mL (0.5 mM)

²                          Samples prepared at a concentration of 388 µg/mL (0.5 mM)

CV        Coefficient of Variation

Table B            Historic Data for Positive Controls

	Cysteine peptide concentration (µg/mL)^1	Lysine peptide concentration (µg/mL)^2
Maximum	107	174
Minimum	101	153
Mean	104^3	162^4
Number	18	18
CV (%)	1.56	3.87

¹                          Samples prepared at a concentration of 376 µg/mL (0.5 mM)

²                          Samples prepared at a concentration of 388 µg/mL (0.5 mM)

³            Overall mean depletion 72.6% (n=18)

⁴            Overall mean depletion 58.8% (n=18)

CV        Coefficient of Variation

Analyzed concentrations and coefficients of variation were calculated using unrounded figures. Data taken from Envigo study HLS1102.

Interpretation of results:

other: Predicted by DPRA not to be a skin sensitizer.

Conclusions:

Solutions of the test item were successfully analyzed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both the Cysteine and Lysine containing synthetic peptides. With no depletion (reactivity) of either peptide in the presence of the test item is therefore predicted by DPRA as negative and not to be a potential skin sensitizer based on this assay.

Executive summary:

The purpose of this study (based on the OECD guideline for the testing of chemicals,In chemicoSkin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of the test item. 

Solutions of the test item were successfully analyzed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both the Cysteine and Lysine containing synthetic peptides. There was no reactivity of the test item with either the Cysteine peptide and the Lysine peptide and therefore this test item is predicted by DPRA not to be a skin sensitizer. 

 

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 28 January 2019 and Experimental completion date: 19 February 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for the Testing of Chemicals: OECD 442E; In Vitro Skin Sensitisation: In Vitro Skin Sensitisation Assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation.

Version / remarks:

Annex I: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), June 2018.

Deviations:

yes

Remarks:

These deviations to the study plan, however, do not affect the validity of the study

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

The OECD TG 442 E 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.

Details on the study design:

Controls for Cytotoxicity Test and h-CLAT.

Medium Control
- Name: Culture medium

Solvent Control for the Test Item
- Name: DMSO (final concentration 0.2%)

Positive Control (h-CLAT)
- Name: DNCB (2,4-dinitrochlorobenzene, CAS No.: 97-00-7, Sigma-Aldrich, Germany) final concentration: 3 and 4 µg/mL, Purity ≥ 99%)
- Solvent: DMSO (final concentration 0.2%), Fisher ScientificTM, Germany, diluted in culture medium.

Solvent Control for the Positive Control (h-CLAT)
- Name: DMSO (Dimethyl sulfoxide, CAS No. 67-68-5, Fisher ScientificTM, Germany) in culture medium, final concentration 0.2%, Purity ≥ 99%.

TEST ITEM PREPARATION
On the day of the experiment (prior to start) test item was prepared in culture medium which formed a stable suspension/dispersion.
As tested by a solubility test, 5000 µg/mL in culture medium (the OECD 442E guideline recommended maximal to be tested test item concentration) was used as highest test item concentration in the first cytotoxicity test.
For the cytotoxicity test (dose finding assay) eight concentrations of the test item were analysed. For this, dilutions were prepared by 1:2 serial dilutions.
Due to high cytotoxicity observed in all test item concentrations of the first cytotoxicity test, the test item concentrations were adjusted for the second and third cytotoxicity test (highest concentration 50µg/mL).

TEST SYSTEM AND SUPPORTING INFORMATION
THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC.
THP-1 cells are used as a surrogate for human myeloid dendritic cells, because the THP-1 cells also show increased CD86 and/or CD54 expression when exposed with sensitisers.

THP-1 Cell Cultures
Stocks of the THP-1 cell line are stored in liquid nitrogen.
The cells are sub-cultured twice weekly.
The cell density should not exceed 1 E 106 cells/mL.
Cells can be used up to two months after thawing (passage number should not exceed 30).
The passage numbers of the used THP-1 cells were 15 and 17 in the cytotoxicity tests and 18 and 20 in the h CLAT for runs 1 and 2, respectively.

Culture Medium
RPMI 1640 Medium, GlutaMAXTM Supplement including 25 mM HEPES, supplemented with 10 % FBS (v/v), 0.05 mM 2 mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate and appropriate antibiotics (100 U/mL of penicillin and 100 µg/mL of streptomycin) is used to culture the cells during the assay.

Preparation and Seeding of THP-1 Cells
On the day of the cytotoxicity or main experiment (h-CLAT) directly before the treatment of the cells, a volume of 500 µL with a cell density of 1.8 - 2  106 THP-1 cells/mL was seeded in each corresponding well of a 24-well flat bottom plate.

Experimental Design and Procedures of the Cytotoxicity Test
Dose Finding Assay (Flow cytometer)
The test item concentrations investigated in the main experiment (h-CLAT) were determined with two cytotoxicity tests. The tests were performed with independent cell cultures on different days.


Treatment of the Cells
The test item dilutions were prepared freshly before each experiment.
Each volume (500 µL) of the dilutions of the test item and culture medium was added to the cells.
The treated THP-1 cells were incubated for 24 ± 0.5 hours. All dose groups were tested in one replicate for each cytotoxicity test. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations.
Each concentration of the test item, culture medium and solvent control were prepared for the 7-AAD staining.


Staining of the Cells
Each test item-treated and not test item treated cells were collected and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 µL of a 7-AAD solution were added in each sample tube.
Flow Cytometry Acquisition (Cytotoxicity Test)
The following acquisition plots were prepared:
- 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
- Histogram plot of the FL-3 channel
The cell viability was measured by gating-out dead cells stained with 7-AAD. A total of 10,000 living cells were analysed.


Flow Cytometry Analysis (Cytotoxicity Test)
The cell viability is shown by the cytometry analysis program (% total) or is calculated according to the following equation:

Cell Viability [%]= (Number of living cells) / (Number of acquired cells) ×100

The CV75 value, a concentration showing 75% of THP-1 cell survival (25% cytotoxicity), is calculated by log-linear interpolation using the following equation:

Log CV75 = (((75-c)×Log (b)- (75-a) ×Log (d)) / (a-c ))

Where:
a is the minimum value of cell viability over 75%
c is the maximum value of cell viability below 75%
b and d are the concentrations showing the value of cell viability a and c respectively


Acceptability of the Cytotoxicity Assay
The cytotoxicity test is considered to be acceptable if it meets the following criteria:
- The cell viability of the medium and solvent control (if the test item is solved in DMSO) should be more than 90%.

Positive control results:

see below

Key result

Run / experiment:

other: 24 hours incubation

Parameter:

other: RFI of CD86 (Relative Fluorescence Intensity) in %.

Remarks:

for the test item

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: prediction is considered positive for the test item in this h-CLAT

Key result

Run / experiment:

other: 24 hours incubation

Parameter:

other: RFI of CD54 (Relative Fluorescence Intensity) in %

Remarks:

for the test item.

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: prediction is considered positive for the test item in this h-CLAT

Other effects / acceptance of results:

Aceptance criteria
The following acceptance criteria should be met when using the h-CLAT method:
·      Cell viability of medium control and DMSO control should be more than 90%.
·      In the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
·      For both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of 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 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50%in at least one concentration of the two tested positive control concentrations.
·      For the test chemical, the cell viability should be more than 50% in at least four tested concentrations in each run.
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).


Prediction model
For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs (OECD 442E guideline):
− The RFI of CD86 is ≥ 150% at any tested concentration (with cell viability ≥ 50%);
− The RFI of CD54 is ≥ 200% at any tested concentration (with cell viability ≥ 50%).
Otherwise, the h-CLAT prediction is considered NEGATIVE (see chapter 5.6.7.1).
Based on the above, if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered POSITIVE and a third run does not need to be conducted. Similarly, if the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE without the need for a third run. If, however, the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 (hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third run is required. If this third run is negative for both markers (hereinafter referred 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 referred 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).

Results of the h-CLAT Test

Results of the first h-CLAT run for the Test Item.

	Concentration (µg/mL)	Antibody / ISO	MFIGeoMean(FITC)	MFI-ISO	RFI (%)	Cell Viability (%)	Cell Viability ISO (%)
Medium control	-	ISO	2.73			95.75	95.75
		CD54	3.35	0.62	100.0	95.29
		CD86	3.72	0.99	100.0	96.07
DMSO control	-	ISO	2.66			96.24	96.24
		CD54	3.45	0.79	100.0	96.34
		CD86	3.69	1.03	100.0	96.09
Positive control (DNCB)	3	ISO	4.02			79.23	79.23
		CD54	6.73	2.71	343.0	79.37
		CD86	24.13	20.11	1952.4	90.88
	4	ISO	4.05			79.75	79.75
		CD54	9.19	5.14	650.6	78.81
		CD86	21.28	17.23	1672.8	87.73
Test Item	1.8	ISO	3.13			95.82	95.82
		CD54	4.10	0.97	156.5	96.07
		CD86	5.92	2.79	281.8	98.09
	2.2	ISO	3.30			95.56	95.56
		CD54	3.98	0.68	109.7	94.92
		CD86	6.42	3.12	315.2	98.27
	2.7	ISO	3.41			95.12	95.12
		CD54	4.19	0.78	125.8	94.72
		CD86	6.89	3.48	351.5	98.24
	3.2	ISO	3.59			92.44	92.44
		CD54	4.46	0.87	140.3	93.79
		CD86	6.81	3.22	325.3	97.11
	3.8	ISO	3.62			92.67	92.67
		CD54	4.75	1.13	182.3	92.66
		CD86	7.05	3.43	346.5	96.63
	4.6	ISO	3.91			86.39	86.39
		CD54	7.47	3.56	574.2	86.41
		CD86	7.86	3.95	399.0	91.87
	5.5	ISO	4.69			67.36	67.36
		CD54	12.97	8.28	1335.5	67.40
		CD86	13.06	8.37	845.5	78.06
	6.6	ISO	5.98			32.80	32.80
		CD54	12.08	6.10	983.9	34.63
		CD86	42.19	36.21	3657.6	51.75

Shaded test groups:          cytotoxic effects occurred by the Flow Cytometric Evaluation (< 50% cell viability)

 

Results of the second h-CLAT run for the Test Item

	Concentration (µg/mL)	Antibody / ISO	MFIGeoMean(FITC)	MFI-ISO	RFI (%)	Cell Viability (%)	Cell Viability ISO (%)
Medium control	-	ISO	2.26			95.35	95.35
		CD54	3.36	1.10	100.0	95.24
		CD86	5.71	3.45	100.0	97.19
DMSO control	-	ISO	2.34			95.35	95.35
		CD54	3.64	1.30	100.0	95.57
		CD86	6.41	4.07	100.0	98.31
Positive control (DNCB)	3	ISO	2.87			84.61	84.61
		CD54	7.97	5.10	392.3	84.98
		CD86	19.20	16.33	401.2	90.03
	4	ISO	2.87			83.93	83.93
		CD54	9.34	6.47	497.7	84.87
		CD86	20.86	17.99	442.0	88.65
Test Item	1.8	ISO	2.63			94.35	94.35
		CD54	3.55	0.92	83.6	94.79
		CD86	6.12	3.49	101.2	97.40
	2.2	ISO	2.67			94.78	94.78
		CD54	3.72	1.05	95.5	94.06
		CD86	5.97	3.30	95.7	96.71
	2.7	ISO	2.71			95.14	95.14
		CD54	3.97	1.26	114.5	94.58
		CD86	7.15	4.44	128.7	97.34
	3.2	ISO	2.93			92.89	92.89
		CD54	4.83	1.90	172.7	93.03
		CD86	6.94	4.01	116.2	95.12
	3.8	ISO	3.30			91.84	91.84
		CD54	6.68	3.38	307.3	92.34
		CD86	7.37	4.07	118.0	93.83
	4.6	ISO	3.63			84.20	84.20
		CD54	11.54	7.91	719.1	85.27
		CD86	8.92	5.29	153.3	88.04
	5.5	ISO	3.93			70.34	70.34
		CD54	16.73	12.80	1163.6	70.62
		CD86	15.31	11.38	329.9	77.22
	6.6	ISO	4.73			41.30	41.30
		CD54	13.94	9.21	837.3	41.82
		CD86	28.36	23.63	684.9	50.24

Shaded test groups:          cytotoxic effects occurred by the Flow Cytometric Evaluation (< 50% cell viability)

Acceptance Criteria of the h-CLAT

Acceptance Criteria of the first h-CLAT run for the Test Item

 

Cell viability of medium control and DMSO control should be more than 90%.            Medium =      95.75%            DMSO =         96.24%

In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability should be > 50%.            3 µg/mL DNCB (CD 54):        343.0%            3 µg/mL DNCB (CD 86):       1952.4%            4 µg/mL DNCB (CD 54):        650.6%            4 µg/mL DNCB (CD 86):       1672.8%

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 should not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).            CD54:   127.4%            CD86:   104.0%

For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control should be > 105%.            Medium CD 54:         122.7%            Medium CD 86:         136.3%            DMSO CD 54:           129.7%            DMSO CD 86:           138.7%

 

Acceptance Criteria ofthe second h-CLAT run for the Test Item

 

Cell viability of medium control and DMSO control should be more than 90%.            Medium =      95.35%            DMSO =         95.35%

In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability should be > 50%.            3 µg/mL DNCB (CD 54):       392.3%            3 µg/mL DNCB (CD 86):       401.2%            4 µg/mL DNCB (CD 54):       497.7%            4 µg/mL DNCB (CD 86):       442.0%

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 should not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).            CD54:   118.2%            CD86:   118.0%

For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control should be > 105%.            Medium CD 54:         148.7%            Medium CD 86:         252.7%            DMSO CD 54:           155.6%            DMSO CD 86:           273.9%

 

Historical Control Data

These values represent historical control data conducted in 2017/2018.

	Positive Control				DMSO- /Medium Control
	DNCB (2 µg/mL)		DNCB (3 µg/mL)		(MFI - MFI(ISO)DMSO)/ (MFI - MFI(ISO)Medium)
Antibody	CD 54	CD 86	CD 54	CD 86	CD 54	CD 86
Value should be	≥ 200%	≥ 150%	≥ 200%	≥ 150%	< 200%	< 150%
Mean Value [%]	271.0	486.5	393.7	595.2	107.2	99.3
Standard Deviation	85.9	206.1	138.4	202.0	15.6	12.6
Max Value [%]	597.8	1241.5	895.4	1466.9	167.2	140.5
Low Value [%]	89.2	132.4	192.0	175.9	66.3	68.6
Total number of runs	114
Number of runs which did not fulfil the acceptance criterion	15	3	2	1^#	0	0

^#not measured

Interpretation of results:

other: The test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Conclusions:

The test item with a log Pow of 3.4 – 5.3 (estimated) activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Executive summary:

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of the test item prepared in culture medium which formed a stable suspension/dispersion when administeredto THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h-CLAT) of the test item was previously calculated by two cytotoxicity tests.

Cytotoxic effects were observed in all test item concentrations of the first cytotoxicity test (highest tested concentration5000µg/mL). After adjustment of the test item concentrations, cytotoxicity effectswere observed starting at a test item concentration of 6.25 µg/mL in the second and 12.5 µg/mL in the third cytotoxicity test up to the highest tested concentration (50 µg/mL; threshold ofcytotoxicity: < 75%). The mean CV75 value of both cytotoxicity tests was calculated as 5.5 µg/mL.

The following concentrations of the test item were tested in the main experiments (h-CLAT): 1.8, 2.2, 2.7, 3.2, 3.8, 4.6, 5.5 and 6.6 µg/mL

The test item with a log Pow of 3.4 – 5.3 (estimated) was tested in 2 independent runs. The RFI of CD86 and CD54 was equal or greater than 150% and 200%, respectively, in at least one concentration of both independent runs. Due to a cell viability < 50% at the highest tested concentration of both h-CLAT runs, the results of this test item concentration were excluded from the evaluation. A dose response could be observed at the highest tested concentrations for CD54 and CD86 in both runs. Therefore the h-CLAT prediction is considered positive for the test item in this h-CLAT.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria(CD54 ≥ 200% and CD86 ≥ 150%)and the cell viability was >50%.

In conclusion, the test item with a log Pow of 3.4 – 5.3 (estimated) activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

 

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

Based on the to results obtained from three in vitro/ in chemico test methods (OECD 442D, OECD 442E, OECD 442C) we used the Defined Approach "2 our of 3" according to the Draft OECD Guideline on Dfined appraoches for skin sensitisation. The prediction for our test item is non sensitiser.The conclusion is that the test material is a non-sensitiser and the data is not sufficient for classification.