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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 09 October 2018 and Experimental completion date: 01 November 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: crystal powder
Details on test material:
- Synonym (Trade Name): ADDITIVE® 9735
- CAS Number: 102-40-9
- Molecular Formula: C10H14O4
- Molecular Weight: 198 g/mol
- Appearance: White crystal powder
- Expiration date: 30 August 2019
- Purity: 100%

In vitro test system

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.

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
A kit (Molecular Probes Vybrant MTT kit V13154) was used to determine cell viability. 1 vial from the kit was reconstituted by adding 1 mL of sterile PBS (Gibco 10010) and vortexed mixed until dissolved to give 5 mg/mL MTT in DPBS. 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 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.

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 5.28 μM and 7.50 μM for test 1 and 2, respectively, which lay within the historical control range for this laboratory. The average induction in the three replicates for cinnamic aldehyde at 64 µM were 10.20 and 7.05 for test 1 and 2, respectively. The result of the first test (10.20) did not meet the acceptance criterion of between 2 and 8, however, there was a clear dose-response and therefore the test was accepted.

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: I max
Run / experiment:
test 1
Value:
2.4
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: The Imax tests was >1.5 fold and statistically significant as compared to the DMSO control.
Key result
Parameter:
other: I max
Run / experiment:
test 2
Value:
2.87
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: The Imax tests was >1.5 fold and statistically significant as compared to the DMSO control.
Key result
Parameter:
other: cellular viability in %
Run / experiment:
test 1
Value:
99.2
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
other: IC30 and IC50 could not be calculated.
Key result
Parameter:
other: cellular viability in %
Run / experiment:
test 2
Value:
96.12
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
other: IC30 and IC50 could not be calculated.
Key result
Parameter:
other: EC 1.5 in µM
Run / experiment:
test 1
Value:
87.42
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
5.28 μM
Key result
Parameter:
other: EC 1.5 in µM
Run / experiment:
test 2
Value:
89.76
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
7.50 μM
Other effects / acceptance of results:
Test item results:
The Imax for the test item was 2.40 in test 1 and 2.87 in test 2.
The Imax for both tests was >1.5 fold and statistically significant as compared to the DMSO control.
The cellular viability did not fall below 99.20% in test 1 and 96.12% in test 2 and therefore the IC30 and IC50 could not be calculated.
The EC1.5 for the test item was 87.42 µM and 89.76 µM for tests 1 and 2, respectively.

Negative solvent results.
The average coefficient of variation of the luminescence reading for the negative solvent control (DMSO) was 8.8% and 6.9% for test 1 and 2, respectively, which met the acceptance criterion of below 20%.

Any other information on results incl. tables

Results for the 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

1.07

1.18

0.97

1.34

1.16

1.17

1.36

1.71

1.72

2.10

2.40

2.25

Statistically significant

No

No

No

No

No

No

No

Yes

Yes

Yes

Yes

Yes

Viability (%)

116.01

105.65

106.45

99.20

100.40

106.45

103.32

104.12

111.63

103.46

107.84

103.32

Imax

2.40

 

EC1.5(µM)

87.42

IC30(µM)

N/A

IC50(µM)

N/A

 

 

Determination criteria for the skin sensitisation potential of the test item

Result

Is the Imax>1.5 fold and statistically significant

Yes

Is the cellular viability >70% at the lowest concentration at the EC1.5determining concentration

Yes

Is the EC1.5value <1000µM

Yes

Is there an apparent overall dose-response for luciferase induction

Yes

KeratinoSens™ prediction

Positive

 

 

  Results for Cinnamic Aldehyde – Test 1

Positive control conc. (µM)

4

8

16

32

64

Mean fold induction

1.36

1.80

2.01

4.40

10.20

Statistically significant

No

Yes

Yes

Yes

Yes

Viability (%)

104.19

104.65

101.99

104.39

94.55

Imax

10.20

 

EC1.5(µM)

5.28

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

No (10.20), however, there is a clear dose-response

Pass

EC1.5of positive control within two standard deviations of the historical mean (1.47 – 47.70)

Yes (5.28)

Pass

CV% of blank values < 20%

Yes (8.8%)

Pass

Results for the 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

0.79

0.89

0.91

1.04

1.08

1.13

1.35

1.70

2.05

2.55

2.69

2.87

Statistically significant

No

No

No

No

No

No

No

Yes

Yes

Yes

Yes

Yes

Viability (%)

111.54

96.12

104.56

100.80

106.99

108.44

113.19

115.16

110.88

117.47

113.25

101.72

Imax

2.87

 

EC1.5(µM)

89.76

IC30(µM)

N/A

IC50(µM)

N/A

 

Determination criteria for the skin sensitisation potential of the test item

Result

Is the Imax>1.5 fold and statistically significant

Yes

Is the cellular viability >70% at the lowest concentration at the EC1.5determining concentration

Yes

Is the EC1.5value <1000µM

Yes

Is there an apparent overall dose-response for luciferase induction

Yes

KeratinoSens™ prediction

Positive

Results for Cinnamic Aldehyde – Test 2

Positive control conc. (µM)

4

8

16

32

64

Mean fold induction

1.29

1.53

1.85

3.06

7.05

Statistically significant

No

Yes

Yes

Yes

Yes

Viability (%)

110.35

107.79

112.46

113.58

105.15

Imax

7.05

 

EC1.5(µM)

7.50

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 (7.05)

Pass

EC1.5of positive control within two standard deviations of the historical mean (1.47 – 47.70)

Yes (7.50)

Pass

CV% of blank values < 20%

Yes (6.9%)

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

Mean

24.58

SD

11.56

Laboratory Historical Data Range (Mean +/- 2xSD)

1.47

to

47.70

Applicant's summary and conclusion

Interpretation of results:
other: test item gave a positive response in the ARE-Nrf2 Luciferase Test (KeratinoSens™)
Conclusions:
It was concluded that the test item gave a positive response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the test item is a skin sensitizer.

Executive summary:

The purpose of this study was to support a predictive, adverse-outcome-pathway evaluation of whether the test item, HER (Resorcinol bis-(2-Hydroxyethyl) ether, 2-[3-(2-hydroxyethoxy)phenoxy]ethanol, is likely to be a skin sensitizer using the ARE-Nrf2 Luciferase Test (KeratinoSens™).

The Imaxfor HER (Resorcinol bis-(2-Hydroxyethyl) ether, 2-[3-(2-hydroxyethoxy)phenoxy]ethanol was 2.40 in test 1 and 2.87 in test 2. The Imaxfor both tests was >1.5 fold and statistically significant as compared to the DMSO control. The cellular viability did not fall below 99.20% in test 1 and 96.12% in test 2 and therefore the IC30and IC50could not be calculated. The EC1.5for HER (Resorcinol bis-(2-Hydroxyethyl) ether, 2-[3-(2-hydroxyethoxy)phenoxy]ethanol was 87.42 µM and 89.76 µM for tests 1 and 2, respectively. Graphs for HER (Resorcinol bis-(2-Hydroxyethyl) ether, 2-[3-(2-hydroxyethoxy)phenoxy]ethanol showed an overall dose-response for luciferase induction.

 

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 EC1.5values of the positive control, cinnamic aldehyde were 5.28 μM and 7.50 μM for test 1 and 2, respectively, which lay within the historical control range for this laboratory. The average induction in the three replicates for cinnamic aldehyde at 64 µM were 10.20 and 7.05 for test 1 and 2, respectively. The result of the first test (10.20) did not meet the acceptance criterion of between 2 and 8, however, there was a clear dose-response and therefore the test was accepted.

The average coefficient of variation of the luminescence reading for the negative solvent control (DMSO) was 8.8% and 6.9% for test 1 and 2, respectively, which met the acceptance criterion of below 20%.

It was concluded that HER (Resorcinol bis-(2-Hydroxyethyl) ether, 2-[3-(2-hydroxyethoxy)phenoxy]ethanol gave a positive response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that HER (Resorcinol bis-(2-Hydroxyethyl) ether, 2-[3-(2-hydroxyethoxy)phenoxy]ethanol is a skin sensitizer.