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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:
weight of evidence
Study period:
2018-01-31 to 2018-07-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
adopted 04 February 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
The induction of the Keap1-Nrf2-ARE signalling pathway by small electrophilic substances such as skin sensitisers was reported by several studies and represents the second key event of the skin sensitisation process as described by the AOP. Therefore, the KeratinoSens™ assay is considered relevant for the assessment of the skin sensitisation potential of chemicals.

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium 2-(1-carboxylatoethoxy)-1-methyl-2-oxoethyl isooctadecanoate
EC Number:
266-533-8
EC Name:
Sodium 2-(1-carboxylatoethoxy)-1-methyl-2-oxoethyl isooctadecanoate
Cas Number:
66988-04-3
Molecular formula:
C24H44O6.Na
IUPAC Name:
sodium 2-(1-carboxylatoethoxy)-1-methyl-2-oxoethyl isooctadecanoate
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch no. of test material: Pationic ISL (Lot: 1731200025)
- Expiration date of the lot/batch: 12 months after opening

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Solubility and stability of the test substance in the solvent/vehicle: All test item solutions were freshly prepared immediately prior to use.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dissolved in tetrahydrofuran (THF, purity ≥99%, CAS No.: 109-99-9, Sigma, Lot No.: STBH3251). A stock solution of 200 mM was prepared by pre-weighing the test material into a glass vial. A stable suspension was formed when diluted 1:100 in cell culture medium.
- Final dilution of a dissolved solid, stock liquid or gel: Based on the THF stock solution serial dilutions were made using the solvent (THF) to obtain 12 master concentrations of the test item (0.098 to 200 mM). The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the master solutions were further diluted 1:25 in cell culture medium.
These 1:25 diluted test item solutions were further diluted 1:4 when added to the cells so that the final concentrations of the test item range from 0.98 to 2000 µM. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and the respective solvent control.

In vitro test system

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

CELL LINE:
The test was carried out using the transgenic cell line KeratinoSens™ (Givaudan, Switzerland), a cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and were used for routine testing. Only cells at a low passage number <25 (P 10 in experiment 1; P 9 in experiment 2) were used.

Cells were cultured in 75 cm^2 culture flasks (Greiner) in maintenance medium at 37 ± 1°C and 5% CO2 in a humidified incubator. For test item exposure, cells were cultured in test item exposure medium.

LUCIFERASE ASSAY SYSTEM:
The luciferase activity was determined using the following products purchased from Promega. All components were used according to the instructions of the manufacturer´s manual.

Luciferase Assay System 10-Pack
The kit (Promega, Cat. No.: E1501, Lot No.: 0000277734) consisted of the following components relevant for this study:
- 10 vials Luciferase Assay Substrate (lyophilised)
- 10 x 10 mL Luciferase Assay Buffer
If freshly prepared, Luciferase Assay Substrate was dissolved in Luciferase Assay Buffer.
If thawed from -80 °C, Luciferase Assay Reagent was equilibrated to room temperature prior to use.

Luciferase Cell Culture Lysis 5x Reagent
The kit (Promega, Cat. No.: E1531, Lot No.: 0000246522) consisted of 30 mL Luciferase Cell Culture Lysis 5x Reagent.
Prior to use lysis buffer was diluted 1:5 with dist. water (Sigma; Lot No.: RNBG3520).

DOSE GROUPS:
Solvent Controls: 1% (v/v) DMSO in test item exposure medium (for the positive control) 1% (v/v) THF in test item exposure medium (for the test item)
Positive Control: cinnamic aldehyde (4 μM; 8 μM; 16 μM; 32 μM; 64 μM)
Test Item: 12 serial concentrations (0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.50, 125, 250, 500, 1000, 2000 μM)

Each concentration step of the test item and the positive control was assessed in three replicates in every independent run. The solvent controls were assessed using six replicates per 96-well plate in every independent run, respectively.

EXPERIMENTAL PROCEDURE:
The incubation was performed in 96-well plates. Cells were counted by Neubauer chamber and a cell suspension of 8 × 10^4 cells/mL in assay medium was prepared. 125 μL of the cell suspension corresponding to 1 × 10^4 cells were dispensed in each well except for the blank. Cells were mixed by swinging during pipetting into the 96-well plate to ensure homogeneous cell number distribution. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability.

After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 μL test item exposure medium. 50 μL of the freshly prepared 25 times-diluted master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item.

All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.

Luciferase activity
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 μL of lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light.

Plates with the cell lysate were placed in the plate reader (Tecan, Infinite 200Pro) for luminescence measurement. For each well 50 μL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1000 ms before assessing the luciferase activity for 2000 ms. This procedure was repeated for each individual well of 96-well plate.

Cell viability
For the cell viability plate the medium was replaced with 200 μL test item exposure medium. 27 μL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 200 μL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5% CO2 overnight. After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm using a plate reader (Tecan, Infinite 200Pro).

DATA ANALYSIS:
For each test item two independent runs using separately prepared test item solutions and independently harvested cells are necessary to derive a prediction. Each independent run consisted of three replicates for every concentration step of the test item and the positive control. In case of discordant results a third independent run is performed.

For every concentration showing >1.5-fold luciferase activity induction, statistical significance (p<0.05) was calculated using a two-tailed Student’s t-test comparing the luminescence values for the three replicated samples with the luminescence values in the solvent control wells.

The lowest concentration with >1.5-fold luciferase activity induction was the value determining the EC1.5 value. It was checked in each case whether this value was below the IC30 value, indicating that there was less than 30% reduction on cellular viability at the EC1.5 determining concentration.

Prediction Model
The KeratinoSens™ prediction for skin sensitisation of the test item is considered positive if the following conditions are met in at least two independently prepared test runs:
- Imax is increased by >1.5-fold and is statistically significant (p<0.05) compared to the solvent control,
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5-fold,
- EC1.5 value is <1000 μM, and
- an apparent overall dose-response relationship for luciferase induction.
If in a given run, all of the three first conditions are met but a clear dose-response relationship for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <1000 μM is considered as inconclusive. A negative result for test items with a log Kow > 7 has to be interpreted with care due to the applicability of the test method.

Results and discussion

Positive control results:
- The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (7.40 in experiment 1; 3.90 in experiment 2).
- The calculated EC1.5 was between 7 and 34 µM (7.72 µM in experiment 1; 12.71 µM in experiment 2).

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: Experiment I at 15.63 µM
Parameter:
other: max luciferase activity (Imax) induction
Value:
1.64
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Run / experiment:
other: Experiment II at 15.63 µM
Parameter:
other: max luciferase activity (Imax) induction
Value:
2.06
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Run / experiment:
other: Experiment I
Parameter:
other: EC1.5 [µM]
Value:
12.42
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Run / experiment:
other: Experiment II
Parameter:
other: EC1.5 [µM]
Value:
7.18
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
The acceptance criteria proposed by the OECD test guideline 442D were met in this test.

For individual results see Table 1 in box 'Any other information on results incl. tables'.

Any other information on results incl. tables

Table 1: Induction of Luciferase Activity - Overall Induction

 

Concentration [µM]

Relative Fold Induction1)

Experiment 1

Experiment 2

Mean

SD

Positive Control

4.00

1.05

1.14

1.09

0.06

8.00

1.53

1.29

1.41

0.17

16.00

1.75

1.65

1.70

0.07

32.00

3.81

2.13

2.97

1.18

64.00

7.40

3.90

5.65

2.47

Test Item

0.98

1.04

1.24

1.14

0.14

1.95

1.14

1.14

1.14

0.00

3.91

1.16

1.18

1.17

0.02

7.81

1.30

1.56

1.43

0.19

15.63

1.64

2.06

1.85

0.30

31.25

0.17

0.00

0.09

0.12

62.50

0.00

0.00

0.00

0.00

125.00

0.00

0.00

0.00

0.00

250.00

0.00

0.00

0.00

0.00

500.00

0.00

0.00

0.00

0.00

1000.00

0.00

0.00

0.00

0.00

2000.00

0.00

0.00

0.00

0.00

1) Percentage of fold induction is relative to the solvent control (i.e. set at 100%).

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
In this study (performed in accordance with OECD 442D) under the given conditions the test item sodium isostearoyl lactylate induced the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, sodium isostearoyl lactylate can be considered as sensitiser in this test. However, based on the adverse outcome pathway (AOP) for skin sensitisation, a positive outcome in only one of the 3 key events is not sufficient for classification according to CLP.
Executive summary:

In a skin sensitisation study conducted according to OECD 442D, the sensitisation potential of the test item sodium isostearoyl lactylate was assessed on the basis of the activation of keratinocytes by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. Cells were incubated with the test item (12 concentrations tested, ranging from 0.98-2000 µM) for 48 h at 37 °C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

 

In the first experiment, a max luciferase activity (Imax) induction of 1.64 was determined at a test item concentration of 15.63 μM. The corresponding cell viability was >70% (115.4%). In the second experiment, a max luciferase activity (Imax) induction of 2.06 was determined at a test item concentration of 15.63 μM. The corresponding cell viability was >70% (107.8%). The lowest tested concentration with a significant luciferase induction >1.5-fold (1.56) was found to be 7.81 μM. The corresponding cell viability was >70% (107.0%). A dose-response relationship for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

 

Under the condition of this study sodium isostearoyl lactylate is therefore considered as sensitiser.

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