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Toxicological information

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:
2018-01-05 to 2018-04-26
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:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
Adopted 04 February 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
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

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
All test item solutions were freshly prepared immediately prior to use. The test item was dissolved in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity ≥99%; AppliChem; Lot No.: 0001055932). A stock solution of 200 mM was prepared by pre-weighing the test material into a glass vial. Vortex mixing was used to aid solubilisation.
Based on the DMSO stock solutions, serial dilutions were made using the solvent to obtain 12 master concentrations of the test item (0.098 to 200 mM) The stock solution of the test item were 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 diluted 1:4 in cell culture medium when incubated with the cells so that the final concentrations of the tested chemical ranged from 0.98 to 2000 µM. Based on this procedure the final concentration of the solvent (DMSO) was 1% (v/v) in all test item concentrations and controls.

In vitro test system

Details on 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 (passage 12, experiment 1; passage 11, experiment 2) were used. Cells were cultured in 75 cm² culture flasks (Greiner) in maintenance medium at 37 +/- 1 °C and 5% CO2 in a humidified incubator. For test material exposure, cells were cultured in 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 manufacture manual. The kit (Promega, Cat. No.: E1501, Lot No.: 0000276369) consisted of the following components relevant for this study:
- 10 vials Luciferase Assay Substrate (lyophilized)
- 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 allowed to equilibrate to room temperature prior to use.
Luciferase Cell Culture Lysis 5x Reagent
The kit (Promega, Cat. No.: E1531, Lot No.: 0000246522) consisted of the following components relevant for this study:
- 30 mL Luciferase Cell Culture Lysis 5x Reagent
Prior to use lysis buffer was diluted 1:5 with dist. water (Sigma; Lot No.: RNBG3519)

DOSE GROUPS:
Negative Control: DMSO: 1% (v/v) in test item exposure medium
Positive Control: Cinnamic aldehyde: 4 µM, 8 µM, 16 µM; 32 µM; 64 µM
Test Item: 12 concentrations of the test item: 0.98 µM, 1.95 µM, 3.91 µM, 7.81 µM, 15.63 µM, 31.25 µM, 62.50 µM, 125.0 µM, 250.0 µM, 500.0 µM, 1000 µM, 2000 µM
Each concentration step of the test item and the positive control was assessed in three replicates in every independent run. The negative control was assessed using six replicates per plate in every independent run.

EXPERIMENTAL PROCEDURE:
The incubation was performed in 96-well plates.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. 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 shortly before 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 96-well 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 (Gibco Life Science; Lot No.: 1877596). Subsequently 20 µL of passive 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 for luminescence measurement. Per well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1.000 ms before assessing the luciferase activity for 2.000 ms. This procedure was repeated for each individual well.

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 (experiment 1 and 2). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.

DATA ANALYSIS:
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 (negative) 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 test item is considered positive in accordance with UN GHS “Category 1” for skin sensitisation if the following conditions were met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p< 0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is <1000 µM
- an apparent overall dose-response for luciferase induction

If in a given repetition, all of the three first conditions are met but a clear dose-response 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.

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 (3.36 in experiment 1; 3.17 in experiment 2).
- The calculated EC1.5 was between 7 and 34 µM (20.65 µM in experiment 1; 14.27 µM in experiment 2).

In vitro / in chemico

Resultsopen allclose all
Parameter:
other: max luciferase activity (lmax) induction
Run / experiment:
Mean of Experiment 1 and 2
Value:
2.7
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: calculated EC1.5 [µM]
Run / experiment:
Mean of Experiment 1 and 2
Value:
7.58
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: max luciferase activity (lmax) induction
Run / experiment:
Experiment 1, at 15.62 uM
Value:
1.86
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: max luciferase activity (lmax) induction
Run / experiment:
Experiment 2, at 31.25 uM
Value:
3.53
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: calculated EC1.5 [uM]
Run / experiment:
Experiment 1, at 15.62 uM
Value:
6.79
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: calculated EC1.5 [uM]
Run / experiment:
Experiment 2, at 31.25 uM
Value:
8.36
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
OTHER EFFECTS:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
- For each test item two independent repetitions 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 (negative) 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.

ACCEPTANCE OF RESULTS:
- Acceptance criteria: The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition.
- Acceptance criteria met for positive control: Yes

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]

Fold Induction

Significance

Experiment 1

Experiment 2

Mean

SD

Solvent Control

-

1.00

1.00

1.00

0.00

Positive Control

4.00

1.03

1.24

1.13

0.15

8.00

1.25

1.17

1.21

0.06

16.00

1.44

1.59

1.52

0.10

*

32.00

1.63

2.06

1.85

0.30

64.00

3.36

3.17

3.27

0.14

*

Test Item

0.98

1.13

1.23

1.18

0.07

1.95

1.17

1.18

1.17

0.01

3.91

1.11

1.21

1.16

0.07

7.81

1.64

1.40

1.52

0.17

*

15.63

1.86

2.84

2.35

0.70

31.25

1.03

3.53

2.28

1.77

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

0.01

0.00

0.01

2000.00

0.00

0.02

0.01

0.02

* = significant induction according to Student’s t-test, p<0.05

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered to be a sensitiser in accordance with UN GHS category 1.
Executive summary:

In a dermal sensitisation study conducted according to OECD 442D with 4,4’-Dimethoxytrityl chloride (purity 100%) in DMSO, the sensitisation potential of the test item was assessed on the basis of the activation of keratinocytes using the in vitro KeratinoSens™. Cells were incubated with the test item for 48 h at 37 °C and later checked for luciferase activity.

Sensitisation was scored by measuring maximum luciferase activity induction (Imax), cell viability and EC1.5. For both experiment I and II, Imax was greater than a 1.5 fold increase, cell viability was greater than 70% and the value for EC1.5 was less than 1000 µM.

Therefore, in this study, the test item 4,4’-Dimethoxytrityl chloride is considered to be a skin sensitiser under UN GHS “Category 1”.