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

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

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.

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

949-812-5

Molecular formula:

C12H27N(C3H6O)n(C2H4O)m, C14H31N(C3H6O)n(C2H4O)m

IUPAC Name:

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

Test material form:

liquid

Details on test material:

Name: XTJ-785, experimental
Lot No.: 9570-2-6738
CAS No.: Not listed
Purity: >92% C1214 alcohol, propoxylated, aminated, ethyoxylated
Expiry Date: No date established

In vitro test system

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.

Results and discussion

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.

In vitro / in chemico

Results

Results 1

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.

Results 2

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.

Results 3

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.

Results 4

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.

Results 5

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

Results 6

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

Results 7

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%.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.