5'-O-[bis(4-methoxyphenyl)benzyl]-2'-deoxy-N-(2-methyl-1-oxopropyl)guanosine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-12-11 to 2018-05-17

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: February 04, 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

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

68892-41-1

Molecular formula:

C35H37N5O7

Test material form:

solid

Specific details on test material used for the study:

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 solution, serial dilutions were made using the solvent (DMSO) 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 finally diluted 1:4 when added to the cells so that the final concentrations of the tested chemical range 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 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 9 in experiment 1; P 11 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 manufacture’s manual.
Luciferase Assay System 10-Pack
The kit (Promega, Cat. No.: E1501, Lot No.: 0000276369) 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 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 30 mL of 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: CA: 4 µM, 8 µM, 16 µM; 32 µM; 64 µM
Test Item: 12 concentrations of the test item
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. Cells were counted by Neubauer chamber. 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 (see section 10.2) 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 (Gibco Life Science; Lot No.: 1909266). 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 1 second before assessing the luciferase activity for 2 seconds. This procedure was repeated for each individual well of the 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 (experiments 1 and 2). After the incubation period the plate was shaken for 10 min and the OD (optical density) 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 (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 are met in at least two independently prepared test runs:
- 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-fold;
- EC1.5 value is < 1000 µM;
- 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 run will be considered inconclusive and further testing may be required. In addition, a negative result obtained with concentrations < 1000 µM will be 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 (4.34 in experiment 1; 4.64 in experiment 2).
- The calculated EC1.5 was between 7 and 34 µM (11.63 µM in experiment 1; 11.54 µM in experiment 2).

In vitro / in chemico

Results

Results 1

Run / experiment:

other: Mean of Experiment I and II at 2000 µM concentration of the test item

Parameter:

other: max luciferase activity (Imax) induction

Value:

2.86

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Run / experiment:

other: Experiment I, at 2000 µM

Parameter:

other: max luciferase activity (Imax) induction

Value:

11.63

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Run / experiment:

other: Experiment II, at 2000 µM

Parameter:

other: max luciferase activity (Imax) induction

Value:

11.54

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 4

Run / experiment:

other: Mean of Experiment I and II

Parameter:

other: calculated EC1.5 [µM]

Value:

11.58

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 5

Key result

Run / experiment:

other: Experiment I

Parameter:

other: calculated EC1.5 [µM]

Value:

11.63

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 6

Key result

Run / experiment:

other: Experiment II

Parameter:

other: calculated EC1.5 [µM]

Value:

11.54

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no 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.

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]	Relative Fold Induction^1)				Significance
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	1.00	1.00	1.00	0.00
Positive Control	4.00	1.23	1.38	1.31	0.11
	8.00	1.20	1.19	1.19	0.01
	16.00	1.55	1.30	1.42	0.18
	32.00	2.13	2.57	2.35	0.31	*
	64.00	4.34	4.64	4.49	0.22	*
Test Item	0.98	1.03	1.06	1.05	0.02
	1.95	1.03	1.12	1.08	0.06
	3.91	1.05	1.22	1.14	0.12
	7.81	1.12	1.18	1.15	0.04
	15.63	1.89	1.85	1.87	0.03	*
	31.25	1.72	1.50	1.61	0.16	*
	62.50	1.70	1.85	1.77	0.11	*
	125.00	1.75	1.12	1.44	0.45
	250.00	2.14	0.99	1.56	0.81
	500.00	2.86	0.82	1.84	1.45
	1000.00	4.37	0.91	2.64	2.44
	2000.00	5.00	0.71	2.86	3.04

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

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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non-sensitiser.

Executive summary:

In a dermal sensitisation study conducted according to OECD 442D with N2-Isobutyryl-5’-O-(4,4’-Dimethoxytrityl)-2’-deoxyguanosine%), the sensitisation potential of the test item was assessed on the basis of the activation of keratinocytes using the in vitro KeratinoSens™ cell line. 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. There was no significant luciferase induction >1.5 fold at concentrations where cell viability exceeds the threshold of 70%. No dose-response relationship for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. Therefore, in this study, the test item is considered to be a non-sensitiser under UN GHS Criteria.