N-benzoyl-5'-O-(p,p'-dimethoxytrityl)-2'-deoxycytidine

Endpoint summary

Description of key information

The skin sensitisation potential of the target substance N4-Benzoyl-5’-O-(4,4’-dimethoxytrityl)-2’-deoxycytidine was assessed in two in vitro skin sensitisation assays (OECD 442D and OECD 442E) and in one in vivo LLNA (OECD 429, read-across).

In the first study conducted according to OECD 442E, the sensitisation potential of the test item was assessed based on the activation of dendritic cells using the in vitro human cell line activation test (h-CLAT). Based on the results, the test item is not considered to be a skin sensitizer. However, in accordance to the OECD testing guideline 442E negative results with test items with a Log Kow greater than 3.5 should not be considered. As the predicted Log Kow of the test item is 5.3 the study will not be used for classification.

In the second study conducted according to OECD 442D (Keratinosens) the sensitisation potential of the test item was assessed based on the activation of keratinocytes. No sensitising potential was assessed in this test for the substance.

To further assess the skin sensitisation potential of the target substance, available in vivo data from a suitable read-across partner was used.

The source substance (5’-O-(4,4’-Dimethoxytrityl)thymidine, CAS 40615-39-2) was tested in an in vivo LLNA study conducted according to OECD 429. None of the tested concentrations exceeded the stimulation index of 3. Therefore, an EC3 value could not be calculated and the test item must be considered as non-sensitizer.

Furthermore, supporting information of the skin sensitisation potential of the target substance was predicted by QSAR, using the Skin Sensitisation models CAESAR 2.1.6 and IRFMN/JRC 1.0.0, which are implemented in the QSAR tool VEGA (core version 1.2.8.). Both QSAR models gave contradictory predictions. In the model CAESAR 2.1.6 the prediction was negative (non-sensitiser) and in the IRFMN/JRC 1.0.0 model the prediction was positive (sensitiser). Based on these results, no clear prediction can be derived from the QSAR modelling. As in both models the substance was not within the applicability domain the results must be considered as not reliable.

As a conclusion based on an assessment of the available data in a weight-of-evidence approach, the test item is considered to be not sensitizing to the skin and no classification for skin sensitisation is warranted.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-11-29 to 2018-05-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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.

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
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 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 in 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.

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 2 in experiment 1; P 6 in experiment 2) were used. Cells were cultured in 75 cm2 culture flasks (Greiner) in maintenance medium at 37 ± 1 °C and 5% CO2. For test item exposure, cells were cultured in medium for test item exposure.

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.: 0000254353; 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: 1% (v/v) DMSO 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: 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. 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 homogenous 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 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 (Gibco Life Science; Lot No.: 1909266; 1877596). 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. 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 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 (experiment 1) or over the weekend (experiment 2). 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 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.

PREDICTION MODEL:
The test item is considered positive in accordance with UN GHS “Category 1” if the following conditions were 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 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.

Positive control results:

- The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (4.35 in experiment 1; 2.72 in experiment 2).
- The calculated EC1.5 was between 7 and 34 µM (13.60 µM in experiment 1; 23.97 µM in experiment 2).

Run / experiment:

other: Mean of Experiment 1 and 2

Parameter:

other: max luciferase activity (Imax) induction

Value:

1.23

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

No significant luciferase induction was observed. Therefore, no EC1.5 value could be calculated.

Key result

Run / experiment:

other: Experiment 1, at 0.98 µM

Parameter:

other: max luciferase activity (Imax) induction

Value:

1.17

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

No significant luciferase induction was observed. Therefore, no EC1.5 value could be calculated.

Key result

Run / experiment:

other: Experiment 2, at 0.98 µM

Parameter:

other: max luciferase activity (Imax) induction

Value:

1.3

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

No significant luciferase induction was observed. Therefore, no EC1.5 value could be calculated.

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:
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'.

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	1.24	1.14	1.19	0.07
	8.00	1.23	1.16	1.19	0.05
	16.00	1.62	1.32	1.47	0.21
	32.00	2.05	1.68	1.87	0.26	*
	64.00	4.35	2.72	3.54	1.15
Test Item	0.98	1.17	1.30	1.23	0.09
	1.95	1.13	1.02	1.07	0.08
	3.91	0.07	0.00	0.04	0.05
	7.81	0.00	0.00	0.00	0.00
	15.63	0.00	0.00	0.00	0.00
	31.25	0.00	0.00	0.00	0.00
	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

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

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 two independent experiment runs. Therefore, the test item can be considered to be a non-sensitiser.

Executive summary:

In a dermal sensitisation study conducted according to OECD 442D with N4-Benzoyl-5’-O-(4,4’-dimethoxytrityl)-2’-deoxycytidine (99.65% purity) in DMSO, 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. For both experiments 1 and 2 no significant luciferase induction was observed within the tested concentration range. Therefore, no EC1.5 value could be calculated. Based on the results, the test item N4-Benzoyl-5’-O-(4,4’-dimethoxytrityl)-2’-deoxycytidine is considered to be a non-sensitiser.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

The chemical structures of the source (5’-O-(4,4’-Dimethoxytrityl)thymidine) and the target substance (N4-Benzoyl-5’-O-(4,4’-dimethoxytrityl)-2’-deoxycytidine) are considered to be sufficiently similar to justify the read-across approach (structural analogue). Source and target substance only differ by the nucleoside group, whereas the source substance contains a thymidine nucleoside and the target substance contains a cytidine nucleoside. The nucleosides, which are the major structural differences in both substances, are considered to have less impact on toxicological endpoints. The toxic effect of skin sensitization is most likely to be triggered by the dimethoxy trityl group. In addition, the source substance is of high purity and its impurities would not negatively influence the validity of the read-across hypothesis. Finally, as indicated in ECHA‘s guidance on QSAR and grouping of chemicals (ECHA Chapter R.6, 2008a), comparison of the water solubility is an important aspect in determining the similarity between compounds for purposes of the read-across strategy. This approach assumes that the extent of water solubility approximates the bioavailability of a substance. The usage of source substances which have a higher water solubility compared to the target substance can be considered as an appropriate worst-case read-across approach for potential adverse effects and is adequately protective. The source substance used for read-across to the target substance showed indeed a higher water solubility, hence the read-across approach is considered as suitable.

Reason / purpose for cross-reference:

read-across source

Positive control results:

α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1 v/v) was used as a positive control. The positive control substance exceeded the stimulation index of 3 confirming the sensitivity and reliability of the experimental technique (see Table 2 in box "Any other information on results").

Key result

Parameter:

SI

Remarks:

mean of five animals

Value:

1.2

Test group / Remarks:

5%

Key result

Parameter:

SI

Remarks:

mean of five animals

Value:

1.2

Test group / Remarks:

10%

Key result

Parameter:

SI

Remarks:

mean of five animals

Value:

1.4

Test group / Remarks:

25%

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
- Please see Table 2 in box "Any other information on results".

EC3 CALCULATION
- The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

CLINICAL OBSERVATIONS:
- No signs of systemic toxicity were observed during the study period. From days 2 to 6, the animals treated with test item concentrations of 10 and 25% showed an erythema of the ear skin (Score 1). Additionally, the animals of all concentrations showed scaly ears on days 4 and 5. On day 6 scaly ears were still observed in animals treated with 25% and in one animal treated with 10%. No deaths occured during the study period. There were no effects observed on body weight.

BODY WEIGHTS:
- The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Results of vehicle and dose selection

Solubility test:

The maximum concentration of test item which could be technically used was a 50% solution in DMF.

Pre-test: Irritation and toxicity test:

Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25 and 50% once daily each on three consecutive days. At the tested concentrations the animals did not show any signs of systemic toxicity. From day 2 to 6, the animals showed a slight to well defined erythema of the ear skin (Score 1 to 2) and scaly ears. Additionally, the animal treated with 50% test item concentration showed loss of fur, visible swelling of the ears and eschar formation (which was noticed on day 6 upon preparation).

Results of the main study

Table 2: Results of the positive control group

Test item (alpha-Hexylcinnamaldehyde) Concentration [%]	Group	Result Stimulation Index
-	background	-
-	background	-
0	1	1.00
5	2	1.68
10	3	1.78
25	4	8.19

Table 3: Results of the main experiment

Test item (DMT-dT) concentration [%]	Group Number	Animal Number	DPM values measured	DPM−BG per animal (2 lymph nodes)	Stimulation Index	Mean DPM per animal (2 lymph nodes)	standard deviation	Stimulation Index
-	-	background 1	16	-	-	-	-	-
-	-	background 2	12	-	-	-	-	-
Vehicle Control Group (DMF)	1	1	1335	1321	-	1963.4	463.3	1.0
	1	2	2107	2093	-
	1	3	2411	2397	-
	1	4	1672	1658	-
	1	5	2362	2348	-
5% DMT-dT	2	6	1156	1142	0.6	2348.2	765.6	1.2
	2	7	2273	2259	1.2
	2	8	2334	2320	1.2
	2	9	2973	2959	1.5
	2	10	3075	3061	1.6
10% DMT-dT	3	11	1461	1447	0.7	2350.6	995.7	1.2
	3	12	2389	2375	1.2
	3	13	1510	1496	0.8
	3	14	2555	2541	1.3
	3	15	3908	3894	2.0
25% DMT-dT	4	16	2529	2515	1.3	2715.6	441.8	1.4
	4	17	3239	3225	1.6
	4	18	2605	2591	1.3
	4	19	3111	3097	1.6
	4	20	2164	2150	1.1

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, in a mouse local lymph node assay, the test item DMT-dT is not to be considered a skin sensitizer under the test conditions of this study.

Executive summary:

In a dermal sensitization study conducted according to OECD 429, five young adult female CBA/CaOlaHsd mice per dose group were dermally exposed to DMT-dT in DMF at concentrations of 5%, 10% and 25% (w/w) by topical application to the dorsum of each ear for three consecutive days and observed until day 6 in a local lymph node assay (LLNA). α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1 v/v) was used as a positive control. The positive control substance exceeded the stimulation index of 3 at the highest dose tested (25%).

No signs of systemic toxicity were observed during the study period after treatment with the test substance. Animals treated with test item concentrations of 10% and 25% showed very slight erythema of the ear skin. In addition, at all concentrations tested, animals showed scaly ears on days 4 and 5. This observation persisted until day 6 in animals treated with 25% and in one animal treated with 10% DMT-dT. A statistically significant or biologically relevant increase in ear weights was not observed in any treatment group in comparison to the vehicle control group. No mortality or effects on body weight occurred during the study. None of the three tested concentrations exceeded a stimulation index of 3 (1.2 (5%), 1.2 (10%) and 1.4 (25%)). As a consequence, an EC3 value could not be calculated. In this study, DMT-dt is not a dermal sensitizer.

This information is used in a read-across approach in the assessment of the target substance.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-11-29 to 2018-05-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 442E

Version / remarks:

adopted: 09 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

The correlation of upregulation of immunologically relevant cell surface markers with the skin sensitising potential of a chemical has been reported and represents the third key event of the skin sensitisation process as described by the AOP for skin sensitisation. This method, which measures the markers of DC activation, using the DC-like cell line THP-1 is considered relevant for the assessment of the skin sensitisation potential of chemicals.

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was freshly prepared immediately prior to use. The test item was soluble in DMSO at a concentration of 500 mg/mL. Vortex mixing was used to aid solubilisation. Stock solutions were prepared by diluting the highest soluble concentration seven times with a constant dilution factor of 1:2. The working stock solutions were prepared by diluting each stock solution 250 times with cell culture medium. In the main experiments no precipitation, turbidity or phase separation was observed when diluted 1:250 in cell culture medium. In the dose finding assays 1 and 2 precipitation and turbidity was observed when diluted 1:250 in cell culture medium. The working stock solutions were applied to the cells by adding equal volumes of each solution to prepared cells, resulting in a further 1:2 dilution of the working solutions. The solvent (DMSO) was present at a constant volume ratio of 0.2% (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.

Details on the study design:

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

CELL LINE:
The test was carried out using THP-1 cells (ATCC® TIB-202^TM), an acute human monocytic leukemic cell line used as a surrogate for DC. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and subcultured at least 2 weeks before they were used in the in vitro h-CLAT test. Cells at passage number (< 30) were used. Cells are routinely passaged every 2-3 days at a density of 0.1–0.2 x 10^6 cells/mL. Cells were cultured in 75 cm² culture flasks (Greiner) in cell culture medium consisting of Roswell Park Memorial Institute medium (RPMI-1640, Gibco Life Science; Cat. No.: 31870-025) supplemented with 10% fetal bovine serum, 25 mM HEPES, L-glutamine, 0.05 mM 2-mercaptoethanol and 100 U/mL penicillin/ 100 µg/mL streptomycin at 37 +/- 1°C and 5% CO2.

PRE-EXPERIMENTS:
Prior to testing, the quality of freshly thawed cell batch was checked by monitoring the doubling time and checking the reactivity towards defined controls. Hereby, DNCB at a final concentration of 4 µg/mL and nickel sulphate (NiSO4) at a final concentration of 100 µg/mL served as positive control while lactic acid (LA) at a final concentration of 1000 µg/mL served as negative control. Cells were accepted when both, DNCB and nickel sulphate produce a positive response for CD86 and CD54, and lactic acid produces a negative response for CD86 and CD54.

SOLVENT FINDING:
Solubility of the test item was determined prior to the main experiment. The test item was dissolved in 0.9% NaCl at a final concentration of 100 mg/mL. Test items not soluble in 0.9% NaCl solution were dissolved in DMSO at a concentration of 500 mg/mL. If the test item was not soluble in DMSO, other solvents (e.g. THF) were used. It was taken care that the test chemical is dissolved or stably dispersed in the chosen solvent and that it does not interfere with the test design. If the test item was not soluble in DMSO or a different organic solvent at 500 mg/mL, the highest soluble concentration was tested by diluting the solution from 500 mg/mL with a constant factor of 1:2 up to a minimal concentration of 1 mg/mL.

EXPERIMENTAL PROCEDURE:
Dose Finding Assay
Starting from 500 mg/mL (dose finding assay 1) and 20 mg/mL (dose finding assay 2) solutions of the test chemicals, eight other stock solutions were prepared by 2-fold serial dilutions using the appropriate solvent (DMSO). These stock solutions were further diluted 250-fold into culture medium (working solutions). The working solutions were finally used for treatment by adding an equal volume of working solution to the volume of THP-1 cell suspension in a 96-well plate to achieve a further 2-fold dilution. For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of 0.1-0.2 x 10^6 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation and were re-suspended in fresh culture medium at a density of 2 x 10^6 cells/mL. Then 500 µL of the cell suspension were seeded into a 24 well flat-bottom plate (1x 10^6 cells/well). The solvent control and the test item working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2.
After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The supernatant was discarded and the remaining cells were washed twice with Dulbecco’s phosphate buffered saline (DPBS) containing 0.1% bovine serum albumin (BSA; i.e. FACS buffer). After washing, cells were re-suspended in 600 µL FACS buffer. 200 µL of the cell suspension were transferred into a FACS tube and stained by using propidium iodide (PI) solution at a final concentration of 0.625 µg/mL. The PI uptake of the cells and therefore cytotoxicity was analysed immediately after the staining procedure by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ > 650 nm. A total of 10000 living (PI negative) cells were acquired and cell viability was calculated for each test concentration. The CV75 value, i.e. the concentration showing 75% cell survival, was calculated by log-linear interpolation. The CV75 value was used to calculate the concentration range of the test item for the main experiment.

CD54 and CD86 EXPRESSION
The test item was dissolved using DMSO as determined in the pre-experiment (dose-finding assay). Based on the concentration of the pre-determined CV75 value 8 concentrations of the test item were defined for the measurement of the surface marker expression, corresponding to 1.2*CV75; CV75; CV75/1.2; CV75/1.2^2; CV75/1.2^3; CV75/1.2^4; CV75/1.2^5; CV75/1.2^6. If the CV75 could not be determined due to insufficient cytotoxicity of the test item in the dose finding assay, the highest soluble concentration of the test item prepared with each solvent was used as starting dose.
The test item was diluted to the concentration corresponding to 500-fold of the 1.2× CV75. Then, 1.2-fold serial dilutions were made using the corresponding solvent to obtain the 8 stock solutions to be tested. The stock solutions were further diluted 250-fold into the culture medium (working solutions). These working solutions were finally used for cell treatment with a further final 2-fold dilution factor.
For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of 0.1–0.2x 10^6 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation (125 x g) and were re-suspended in fresh culture medium at a density of 2x 10^6 cells/mL. Then 500 µL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 10^6 cells/well). The solvent control, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2.
After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The following steps were carried out on ice with pre-cooled buffers and solutions. The supernatant was discarded and the remaining cells were washed twice with FACS buffer. After washing, cells were blocked using 600 µL of a FcR blocking buffer (FACS buffer containing 0.01% (w/v) Globulin Cohn Fraction) and incubated at 4 °C for 15 min. After blocking, cells were split in three aliquots into a 96-well V-bottom plate. After centrifugation (approx. 250 x g), cells were stained with 50 µL of FITC-labelled anti-CD86, FITC-labelled anti-CD54, or FITC-labelled mouse IgG1 (isotype) antibodies in the dark for 30 min at 4 °C. All antibodies were diluted in FACS buffer at an appropriate manner. After washing with FACS buffer two times, cells were re-suspended in FACS buffer and PI solution was added. PI staining was done just prior to the measurement by adding PI solutions to each sample (final concentration of PI was 0.625 µg/mL). The expression levels of CD86 and CD54 as well as cell viability (as determined by living cells with no PI uptake) were analysed by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ= 530 nm ± 15 nm for FITC and λ > 650 nm for PI. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 were calculated. The cell viability was calculated.

DATA ANALYSIS:
FACS data analysis was performed using the software BD FACS DIVA 6.0. Further data analysis like calculation of the CV75, calculation of the RFI and calculation of the Effective Concentration 150 and Effective Concentration 200 values were performed using the software Microsoft Excel 2010. The mean values and standard deviations of the single replicates were determined using the respective excel commands.

EVALUATION of RESULTS:
For CD86/CD54 expression measurement, the test item was tested in at least two independent runs to derive a single prediction. Each independent run was performed on a different day or on the same day provided, that for each run, independent fresh stock solutions and working solutions of the test chemicals and antibody solutions were prepared and independently harvested cells were used. Sensitising potential of the test item was predicted from the mean percentage expression of CD86 and CD54. Any test chemical tested by the h-CLAT is considered positive if any of the three scenarios are met:
- the RFI of CD86 is equal to or greater than 150% at any tested dose at a cell viability ≥ 50% in at least two independent runs;
- the RFI of CD54 is equal to or greater than 200% at any tested dose at a cell viability ≥ 50% in at least two independent run;
- the RFIs of both the CD86 and CD54 are equal to or are greater than 150% and 200% respectively at any tested dose at a cell viability ≥ 50% in at least two independent runs.
In case of non-concordant results a third run should be conducted to make the final prediction. Otherwise the result was considered as inconclusive.
A negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x CV75 is < 90%. In contrast, a positive test outcome was accepted irrespective of cell viabilities > 90% at a concentration of 1.2 x CV75. If no CV75 could be derived negative test results can be accepted when the test item is tested at the highest soluble concentration (i.e. 5000 µg/mL for 0.9% NaCl; 1000 µg/mL for DMSO or another organic solvent) even if the cell viability is > 90%. A negative result for test items with a Log KOW > 3.5 should be considered as inconclusive.

Positive control results:

The positive control (DNCB) led to an upregulation of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (262% experiment 1; 329% experiment 2) and 200% for CD54 (285% experiment 1; 363% experiment 2) were clearly exceeded.

Key result

Run / experiment:

other: 1

Parameter:

other: CD86 upregulation

Remarks:

at highest tested concentration of 12.72 µg/mL

Value:

123

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 1

Parameter:

other: CD54 upregulation

Remarks:

at highest tested concentration of 12.72 µg/mL

Value:

115

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: CD86 upregulation

Remarks:

at highest tested concentration of 12.72 µg/mL

Value:

119

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: CD54 upregulation

Remarks:

at highest tested concentration of 12.72 µg/mL

Value:

117

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Summary of Results:

In the present study N4-Benzoyl-5’-O-(4,4’-dimethoxytrityl)-2’-deoxycytidine was dissolved in DMSO. For the dose finding assay stock solutions with concentrations ranging from 3.91 mg/mL to 500 mg/mL (dose finding assay 1, applied concentration range of 7.81 to 1000 µg/mL) and from 0.16 mg/mL to 20 mg/mL (dose finding assay 2, applied concentration range of 0.31 to 40 µg/mL) were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37 °C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis. A CV75 of 10.60 ± 0.95 µg/mL was derived in the dose finding assay. Based on the CV75, the main experiment was performed covering the following concentration steps: 3.55, 4.26, 5.11, 6.13, 7.36, 8.83, 10.60, 12.72 µg/mL. In the main experiments no precipitation or turbidity of the test item was observed for all concentration steps when mixing the test item stock solutions with cell culture medium. In the dose finding assay 1 and 2 precipitation and turbidity of the test item was observed when mixing the test item stock solutions with cell culture medium. Cells were incubated with the test item for 24 h at 37 °C. After exposure cells were labelled with CD54 and CD86 fluorescent antibodies and the expression levels of CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining. Cytotoxicity was observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 73.2% (from the CD86 expression experiment), 72.2% (from the CD54 expression experiment) and 72.8% (when tested withisotype IgG1 control) compared to total number of acquired cells in the first experiment and to 82.3% (CD86), 82.7% (CD54) and 80.7% (isotype IgG1 control) compared to total number of acquired cells in the second experiment. The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% relative to solvent control in any of the experiments. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% relative to solvent control in any of the experiments. Therefore, the test item is not considered to be a skin sensitiser. The controls confirmed the validity of the study for all experiments

Table 1: CD54 and CD86 Expression Experiment 1

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			Isotype IgG1 corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	95.6	94.9	95.0	4282	1626	628	3654	998	81	94	682	259
Solvent Control	0.20%	93.6	94.5	94.4	5133	1696	633	4500	1063	100	100	811	268
DNCB	4.00	82.1	83.4	82.4	12403	3636	603	11800	3033	262	285	2057	603
N4-Benzoyl-5’-O-(4,4’-dimethoxytrityl)-2’-deoxycytidine	12.72	73.2	72.2	72.8	6249	1924	703	5546	1221	123	115	889	274

 

Table 2: CD54 and CD86 Expression Experiment 2

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	IgG Isotype	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	C86	CD54
Medium Control	-	96.8	97.2	97.1	3820	1399	747	3073	652	96	106	511	187
Solvent Control	0.20%	96.9	96.6	96.4	3934	1356	739	3195	617	100	100	532	183
DNCB	4.0	81.9	81.5	80.4	11294	3014	777	10517	2237	329	363	1454	388
N4-Benzoyl-5’-O-(4,4’-dimethoxytrityl)-2’-deoxycytidine	12.72	82.3	82.7	80.7	4566	1494	774	3792	720	119	117	590	193

 

Interpretation of results:

study cannot be used for classification

Conclusions:

In this study under the given conditions the test item N4-Benzoyl-5’-O-(4,4’-dimethoxytrityl)-2’-deoxycytidine did not upregulate the cell surface markers in two independent experiment runs. Based on these results, the test item is not considered to be a skin sensitizer. In accordance to the OECD testing guideline 442E negative results with test items with a logKow greater than 3.5 should not be considered. As the predicted LogKow of the test item is 5.3 the study cannot be used for classifcation.

Executive summary:

In a skin sensitisation study conducted according to OECD 442E with N4-Benzoyl-5’-O-(4,4’-dimethoxytrityl)-2’-deoxycytidine (99.65% purity), the sensitisation potential of the test item was assessed on the basis of the activation of dendritic cells using the in vitro human cell line activation test (h-CLAT). Cells were incubated with the test item for 24 h at 37 °C and later checked for cell viability and expression of CD86 and CD54 cell surface markers. Sensitisation was scored by measuring cell viability and checking the expression of both cell surface markers CD54 and CD86 were not upregulated above the threshold of 200% (CD54) and 150% (CD86) in both experiments. Based on these results, the test item is not considered to be a skin sensitizer under the UN GHS criteria.

In accordance to the OECD testing guideline 442E negative results with test items with a logKow greater than 3.5 should not be considered. As the predicted LogKow of the test item is 5.3 the study cannot be used for classifcation.

Reference 4

Endpoint:

skin sensitisation, other

Remarks:

in silico

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

2019-07-01

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1a. SOFTWARE
VEGA QSAR Models

2a. MODEL (incl. version number)
Skin Sensitization model (CAESAR) 2.1.6
Core version: 1.2.8

3a. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
Compound SMILES: O=C1N=C(C=CN1C5OC(COC(c2ccccc2)(c3ccc(OC)cc3)c4ccc(OC)cc4)C(O)C5)NC(=O)c6ccccc6

4a. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
[Explain how the model fulfils the OECD principles for (Q)SAR model validation. Consider attaching the QMRF or providing a link]
- Defined endpoint: skin sensitization activity

5a. APPLICABILITY DOMAIN
Global AD Index
AD index = 0
Explanation: the predicted compound is outside the Applicability Domain of the model.

Similar molecules with known experimental value
Similarity index = 0.672
Explanation: only moderately similar compounds with known experimental value in the training set have been found.

Accuracy of prediction for similar molecules
Accuracy index = 1
Explanation: accuracy of prediction for similar molecules found in the training set is good.

Concordance for similar molecules
Concordance index = 0
Explanation: similar molecules found in the training set have experimental values that disagree with the predicted value.

Model's descriptors range check
Descriptors range check = False
Explanation: 1 descriptor(s) for this compound have values outside the descriptor range of the compounds of the training set.

Atom Centered Fragments similarity check
ACF index= 0.28
Explanation: a prominent number of atom centered fragments of the compound have not been found in the compounds of the training set or are rare fragments (3 unknown fragments and 3 infrequent fragments found).

1b. SOFTWARE
VEGA QSAR Models (Core Version 1.2.8)

2b. MODEL (incl. version number)
Skin Sensitization model IRFMN/JRC 1.0.0

3b. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
Compound SMILES: O=C1N=C(C=CN1C5OC(COC(c2ccccc2)(c3ccc(OC)cc3)c4ccc(OC)cc4)C(O)C5)NC(=O)c6ccccc6

4b. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
[Explain how the model fulfils the OECD principles for (Q)SAR model validation. Consider attaching the QMRF or providing a link]
- Defined endpoint: skin sensitization activity

5b. APPLICABILITY DOMAIN
Global AD Index
AD index = 0.287
Explanation: the predicted compound is outside the Applicability Domain of the model.

Similar molecules with known experimental value
Similarity index = 0.712
Explanation: only moderately similar compounds with known experimental value in the training set have been found.

Accuracy of prediction for similar molecules
Accuracy index = 1
Explanation: accuracy of prediction for similar molecules found in the training set is good.

Concordance for similar molecules
Concordance index = 1
Explanation: similar molecules found in the training set have experimental values that agree with the predicted value.

Model's descriptors range check
Descriptors range check = True
Explanation: descriptors for this compound have values inside the descriptor range of the compounds of the training set.

Atom Centered Fragments similarity check
ACF index= 0.34
Explanation: a prominent number of atom centered fragments of the compound have not been found in the compounds of the training set or are rare fragments (3 unknown fragments and 2 infrequent fragments found).

Interpretation of results:

other: equivocal results

Conclusions:

The test item is predicted on the one hand to be a non-sensitizer in the VEGA QSAR Model CAESAR 2.6.1 and on the other hand predicted to be a sensitizer in the VEGA QSAR Model IRFMN/JRC 1.0.0. Based on these results, no clear prediction can be derived from the QSAR modelling and the results need to be evaluated as equivocal.

Executive summary:

The test item was predicted as NON-Sensitizer in the VEGA QSAR model CAESAR 2.6.1. Even though there were only moderately similar compounds with known experimental value in the training set (Similarity index = 0.672), and similar molecules found in the training set had experimental values that disagree with the predicted value and one descriptor for this compound had values outside the descriptor range of the compounds of the training set, the accuracy of prediction for similar molecules found in the training set was considered good.

In the VEGA QSAR model IRFMN/JRC 1.0.0 the test item was predicted as Sensitizer. Even though there were only moderately similar compounds with known experimental value in the training set (Similarity index = 0.712), similar molecules were found in the training set which had experimental values that agree with the predicted value, descriptors for this compound had values inside the descriptor range of the compounds of the training set and the accuracy of prediction for similar molecules found in the training set was considered good.

Overall, the QSAR prediction does not allow to draw a conclusion on the sensitizing potential of the test item and the prediction from the both models need to be evaluated as equivocal.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available results and in accordance with CLP regulation 1272/2008, classification of the test item for skin sensitisation is not warranted.