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Administrative data

Description of key information

Two in vitro testing (OECD 442 D and OECD 442 E ) were performed and also one in Chemico study (OECD 442C).

Two of the three studies were negative for skin sensitization (OECD 442C and OECD 442D).  Therefore, the material has not to be classified as skin sensitizer and no LLNA study has to be done for the final classification because of the negative responses in two studies.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

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:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Details on 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.
Skin sensitisation (In vitro test system) - Details on study design:
Placeholder - no text template available yet
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
Parameter:
other: I max for test item
Run / experiment:
test 1
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
Parameter:
other: I max for test item
Run / experiment:
test 2
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
Parameter:
other: cellular viability in %
Run / experiment:
test 1
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
Parameter:
other: cellular viability in %
Run / experiment:
test 2
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
Parameter:
other: IC30 in µM
Run / experiment:
test 1 and test 2
Value:
2.49
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Parameter:
other: IC50 in µM
Run / experiment:
test 1
Value:
2.89
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Parameter:
other: IC50 in µM
Run / experiment:
Test 2
Value:
2.92
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
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 XTJ-785, Experimental – 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 Imaxvalue of 0.70 occurred at a cytotoxic concentration, it was not considered valid. The Imaxvalue 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 XTJ-785, Experimental – 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: not a skin sensitizer
Conclusions:
It was concluded that Alcohols, C12-14 alkyl ethers, propoxylated, aminated, ethoxylated (XTJ-785, Experimental) gave a negative response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that Alcohols, C12-14 alkyl ethers, propoxylated, aminated, ethoxylated (XTJ-785, Experimental) is not a skin sensitizer.

Executive summary:

The purpose of this study was to support a predictive, adverse-outcome-pathway evaluation of whether the test item, Alcohols, C12-14 alkyl ethers, propoxylated, aminated, ethoxylated (XTJ-785, Experimental), is likely to be a skin sensitizer using the ARE-Nrf2 Luciferase Test (KeratinoSens™).

The Imaxfor Alcohols, C12-14 alkyl ethers, propoxylated, aminated, ethoxylated (XTJ-785, Experimental) was 0.69 in test 1 and 0.94 in test 2. The Imaxfor both tests was <1.5 fold compared to the DMSO control and therefore the EC1.5could 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 Alcohols, C12-14 alkyl ethers, propoxylated, aminated, ethoxylated (XTJ-785, Experimental) showed no overall dose-response for luciferase induction. 

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

It was concluded that Alcohols, C12-14 alkyl ethers, propoxylated, aminated, ethoxylated (XTJ-785, Experimental) gave a negative response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that Alcohols, C12-14 alkyl ethers, propoxylated, aminated, ethoxylated (XTJ-785, Experimental) is not a skin sensitizer.

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:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
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 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 Alcohols, C12-14 alkyl ethers, propoxylated, aminated, ethoxylated (XTJ 785, Experimental) 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 Alcohols, C12-14 alkyl ethers, propoxylated, aminated, ethoxylated (XTJ-785, Experimental) 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 XTJ-785, Experimental 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 XTJ-785, Experimental. 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 XTJ-785, Experimental 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 XTJ-785, Experimental. For the co-elution control, buffer solution was used in place of the Lysine stock solution.

Incubation
The appearance of XTJ-785, Experimental 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 XTJ-785, Experimental 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
Parameter:
other: Mean Cysteine depletion in the presence of the test item
Run / experiment:
achieved results
Value:
-1.95
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
72.9% (positive comtrol depletion)
Key result
Parameter:
other: Mean Lysine depletion in the presence of the test item
Run / experiment:
achieved results
Value:
-2.33
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
58.3% (positive control depletion)

Solubility Assessment

Solubility of Alcohols, C12-14 alkyl ethers, propoxylated, aminated, ethoxylated (XTJ-785, Experimental) 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

r2>0.99

60.8-100
(SD <14.9%)

0.45-0.55 mM (CV <15%)

SD <14.9%

Lysine

r2>0.99

40.2-69.0
(SD <11.6%)

0.45-0.55 mM (CV <15%)

SD <11.6%

Achieved results

Cysteine

r2>0.999

72.9
(SD, 0.06%, n=3)

B: 0.510 mM (CV 0.36%, n=6)

SD 0.13% (n=3)

Lysine

r2>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 Alcohols, C12-14 alkyl ethers, propoxylated, aminated, ethoxylated (XTJ-785, Experimental) 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 Alcohols, C12-14 alkyl ethers, propoxylated, aminated, ethoxylated (XTJ-785, Experimental) 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

1043

1624

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:
GHS criteria not met
Remarks:
Predicted by DPRA not to be a skin sensitizer. 
Conclusions:
Solutions of Alcohols, C12-14 alkyl ethers, propoxylated, aminated, ethoxylated (XTJ-785, Experimental) 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, Alcohols, C12-14 alkyl ethers, propoxylated, aminated, ethoxylated (XTJ-785, Experimental) 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 Alcohols, C12-14 alkyl ethers, propoxylated, aminated, ethoxylated (XTJ-785, Experimental). 

Solutions ofAlcohols, C12-14 alkyl ethers, propoxylated, aminated, ethoxylated (XTJ-785, Experimental)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 Alcohols, C12-14 alkyl ethers, propoxylated, aminated, ethoxylated (XTJ-785, Experimental) with either the Cysteine peptide and the Lysine peptide and therefore this test item is predicted by DPRA not to be a skin sensitizer. 

 

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:
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. certificate)
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 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) XTJ-785 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
Parameter:
other: RFI of CD86 (Relative Fluorescence Intensity) in %.
Remarks:
for the test item
Run / experiment:
24 hours incubation
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
Parameter:
other: RFI of CD54 (Relative Fluorescence Intensity) in %
Remarks:
for the test item.
Run / experiment:
24 hours incubation
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 ofthe first h-CLAT run for the Test Item XTJ-785

 

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 XTJ-785

 

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 XTJ-785 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) ofXTJ-785prepared 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 XTJ-785 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 as5.5 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 XTJ-785 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)

Justification for classification or non-classification

According the to results obtained in all three in vitro (OECD 442D and OECD 442E) and in Chemico studies (OECD 442C), the test item can not be classified as skin sensitising based on GHS criteria and no LLNA study need to be performed.