5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine-3′-O-[O-(2-cyanoethyl)-N,N′-diisopropylphosphoramidite]

Endpoint summary

Description of key information

The skin sensitisation potential of the target substance 5′-O-[bis(4-methoxyphenyl)phenylmethyl]-2′-deoxythymidine, 3′-[2-cyanoethyl N,N-bis(1-methylethyl)phosphoramidite] was assessed in two in vitro skin sensitisation assays (OECD 442D and OECD 442E), one in chemico assay (OECD 442C) and one in vivo LLNA (OECD 429).

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 considered to be a skin sensitizer.

In the second study, conducted according to OECD 442D the sensitisation potential of the test item was assessed based on the activation of keratinocytes. Based on the results, the test item is not considered to be a skin sensitizer.

In the third study, conducted according to OECD 442C, the test item showed minimal reactivity towards both peptides. Due to the observed precipitation and phase separation the prediction model does not apply, and a prediction of the sensitizing potential cannot be made.

In addition to the contradictory in vitro/in chemico data and to further assess the skin sensitisation potential of the target substance, available data from an in vivo LLNA study conducted according to OECD 429 is used.

In this LLNA study, none of the tested concentrations exceeded the stimulation index of 3. As a consequence, an EC3 value could not be calculated and the target substance 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-sensitizer) and in the IRFMN/JRC 1.0.0 model the prediction was positive (sensitizer). 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:

2017-12-11 to 2018-05-02

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. 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 dose-finding assays precipitates and turbidity was observed when diluted 1:250 in cell culture medium for the four highest test item concentrations. Sonication was used to aid solubilisation. 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-202TM), 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 106 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.

DOSE GROUPS
- Medium Control: cell culture medium
- Solvent Control: 0.2% DMSO (v/v) in cell culture medium
- Positive Control: 4 µg/mL DNCB
- Test Item:
dose finding assay 1 and 2: 7.81, 15.63, 31.25, 62.50, 125.0, 250.0, 500.0, 1000 µg/mL
main experiment 1, 2 and 3: 11.24, 13.49, 16.19, 19.43, 23.32, 27.98, 33.58, 40.29 µg/mL

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 (NiSO4) produce a positive response and lactic acid (LA) 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 solutions of the test chemicals, eight stock solutions (eight concentrations) were prepared by 2-fold serial dilutions using the corresponding solvent. 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 onto a 24 well flat-bottom plate (1 x 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 – 0.2 x 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 2 x 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 all three experiments. The threshold of 150% for CD86 (378% experiment 1; 281% experiment 2, 327% experiment 3) and 200% for CD54 (527% experiment 1; 285% experiment 2, 341% experiment 3) were clearly exceeded.

Key result

Run / experiment:

other: 1

Parameter:

other: max. CD86 upregulation

Remarks:

at the concentration of 19.43 µg/mL

Value:

188

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 1

Parameter:

other: max. CD54 upregulation

Remarks:

at the concentration of 27.98 μg/mL

Value:

1 374

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: max. CD86 upregulation

Remarks:

at the concentration of 23.32 μg/mL

Value:

97

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: max. CD54 upregulation

Remarks:

at the concentration of 23.32 μg/mL

Value:

84

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: 3

Parameter:

other: max. CD86 upregulation

Remarks:

at the concentration of 27.98 µg/mL

Value:

190

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 3

Parameter:

other: max. CD54 upregulation

Remarks:

at the concentration of 33.58 µg/mL

Value:

351

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive 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:

The in vitro human Cell Line Activation Test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP) for skin sensitisation, namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers after exposure to the test item compared to that of the respective vehicle control is used to support discrimination between skin sensitisers and non-sensitisers.

In the present study 5’-O-[bis(4-methoxyphenyl)phenylmethyl]-2’-deoxythymidine, 3’-[2-cyanoethyl N,N-bis(1-methylethyl)phosphoramidite] was dissolved in dimethyl sulfoxide (DMSO). A CV75 of 33.58 ± 2.29 µg/mL was derived in the dose finding assay. Based on the CV75, the main experiment was performed covering the following concentration steps: 11.24, 13.49, 16.19, 19.43, 23.32, 27.98, 33.58, 40.29 µg/mL. 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. Clear cytotoxicity was observed for the cells treated with the test item in experiment 1 and 3. Relative cell viability at the highest test item concentration was reduced to 51.3% (from the CD86 expression experiment), 50.6% (from the CD54 expression experiment) and 51.2% (when tested with isotype IgG1 control) compared to total number of acquired cells in the first experiment and to 42.3% (CD86), 36.2% (CD54) and 32.1% (isotype IgG1 control) compared to total number of acquired cells in the third experiment. No cytotoxicity was observed in the second experiment. Here, relative cell viability at the highest test item concentration was 96.5% (CD86), 96.4% (CD54) and 95.9% (isotype IgG1 control) compared to total number of acquired cells. In the first experiment the expression of the cell surface marker CD86 was upregulated above the threshold of 150% to a maximum of 188% at a test item concentration of 19.43 µg/mL. At this concentration the cell viability was >50% (82.1%). The expression of the cell surface marker CD54 was upregulated above the threshold of 200% to a maximum of 1374% at a test item concentration of 27.98 µg/mL. At this concentration the cell viability was >50% (70.6%). In the second experiment the expression of the cell surface marker CD86 was not upregulated above the threshold of 150% and the expression of the cell surface marker CD54 was not upregulated above the threshold of 200%. In the third experiment the expression of the cell surface marker CD86 was upregulated above the threshold of 150% to a maximum of 190% at a test item concentration of 27.98 µg/mL. At this concentration the cell viability was >50% (65.6%). The expression of the cell surface marker CD54 was upregulated above the threshold of 200% to a maximum of 351% at a test item concentration of 33.58 µg/mL. At this concentration the cell viability was <50% (46.0%). Thelowest tested concentration with an upregulation above the threshold of 200% (242%) was 23.32 µg/mL.At this concentration the cell viability was >50% (73.4%). The EC150 value was calculated with 70.74 µg/mL and the EC200 value was calculated with 67.64 µg/mL. Since only two independent runs showed an upregulation above the threshold, the higher EC150 or EC200 of the two calculated values was adopted.

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on the results from this in vitro skin sensitisation assay conducted in accordance with OECD 442E, the test item must considered to be a skin sensitiser.

Executive summary:

In a skin sensitisation study conducted according to OECD 442E with 5’-O-[bis(4-methoxyphenyl)phenylmethyl]-2’-deoxythymidine, 3’-[2-cyanoethyl N,N-bis(1-methylethyl)phosphoramidite] (99.7% 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. In the first experiment the expression of the cell surface marker CD86 was upregulated above the threshold of 150% to a maximum of 188% at a test item concentration of 19.43 µg/mL. At this concentration the cell viability was >50% (82.1%). The expression of the cell surface marker CD54 was upregulated above the threshold of 200% to a maximum of 1374% at a test item concentration of 27.98 µg/mL. At this concentration the cell viability was >50% (70.6%).

In the second experiment the expression of the cell surface marker CD86 was not upregulated above the threshold of 150% and the expression of the cell surface marker CD54 was not upregulated above the threshold of 200%.

In the third experiment the expression of the cell surface marker CD86 was upregulated above the threshold of 150% to a maximum of 190% at a test item concentration of 27.98 µg/mL. At this concentration the cell viability was >50% (65.6%). The expression of the cell surface marker CD54 was upregulated above the threshold of 200% to a maximum of 351% at a test item concentration of 33.58 µg/mL. At this concentration the cell viability was <50% (46.0%). Thelowest tested concentration with an upregulation above the threshold of 200% (242%) was 23.32 µg/mL. At this concentration the cell viability was >50% (73.4%). Since both of the cell surface markers clearly exceeded the threshold in at least two independent experiments, the test item can be considered to be a skin sensitiser. 

 

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-12-11 to 2018-05-16

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: March 09, 2018

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
- 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, 0001179895, Honeywell; Lot No.: SZBG293OH). A stock solution of 200 mM was prepared for experiment 1 and 2. During the third experiment a stock solution of 1.5 mM was prepared.
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 for experiment 1 and 2 and 0.0635 to 1.5 mM for experiment 3). The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2 for experiment 1 and 2 and a constant dilution factor of 1:1.333 for experiment 3. 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 for experiment 1 and 2 and from 0.635 to 15 µM for experiment 3. 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 (passage 09 in experiment 1; passage 11 in experiment 2; passage 07 in experiment 3) 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; 0000284055) consisted of the following components relevant for this study:
- 10 vials Luciferase Assay Substrate (lyophilizsed)
- 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.: 0000246922; 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; RNBG3520).

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:
Experiment 1 and 2: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000,
2000 µM
Experiment 3: 0.635, 0.847, 1.13, 1.50, 2.01, 2.67, 3.56, 4.75, 6.33, 8.44, 11.3,
15.0 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 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 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 (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.

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) or over the weekend (experiment 3). 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” for skin sensitisation 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
- EC1.5 value is <1000 µM;
- an apparent overall dose-response relationship for luciferase induction.

If in a given repetition, 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 positive control led to an increase in luciferase activity induction in all the experiments. The threshold of 1.5 (4.34 experiment 1; 4.64 experiment 2; 2.36 experiment 3) was clearly exceeded at the highest concentration of 64 µM.

Key result

Run / experiment:

other: Experiment 1, at 7.81 µM

Parameter:

other: max luciferase activity (Imax) induction

Value:

2.29

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 2, at 15.63 µM

Parameter:

other: max luciferase activity (Imax) induction

Value:

1.62

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Experiment 3, at 11.25 µM

Parameter:

other: max luciferase activity (Imax) induction

Value:

1.78

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Mean of Experiments 1,2 and 3

Parameter:

other: max luciferase (imax) induction

Value:

1.9

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:

In the first experiment, a max luciferase activity (Imax) induction of 2.29 was determined at a test item concentration of 7.81 µM. The corresponding cell viability was >70% (95.7%). No further significant luciferase induction >1.5 was found in the lower concentrations with a viability >70%. Microscopically, cytotoxicity was observed starting from a concentration of 7.81 µM onwards. The calculated EC1.5 was <1000 µM (4.86 µM).
In the second experiment, a max luciferase activity (Imax) induction of 1.62 was determined at a test item concentration of 15.63 µM. The corresponding cell viability was <70% (10.6%). The lowest tested concentration with a luciferase induction >1.5 (1.57) was found to be 7.81 µM. The corresponding cell viability was >70% (95.8%). Microscopically, cytotoxicity was observed starting from a concentration of 7.81 µM onwards. The calculated EC1.5 was <1000 µM (7.47 µM).
Since the test item induced the gene activity very close to the cytotoxic levels, a third experiment with an adapted concentration range and more narrow dose-response analysis with dilution of 1.333-fold between wells instead of two-fold dilutions was performed to decide if induction is at cytotoxic levels or not.
In the third experiment, a max luciferase activity (Imax) induction of 1.78 was determined at a test item concentration of 11.25 µM. The corresponding cell viability was >70% (72.2%). No further significant luciferase induction >1.5 was found in the lower concentrations with a viability >70%. Microscopically, cytotoxicity was observed at the two highest test item concentrations (11.3 and 15.0 µM). The calculated EC1.5 was <1000 µM (9.55 µM).
Within all three experiments the induced the gene activity was very close to the cytotoxic levels. However, no dose-response relationship for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. Therefore, induction of luciferase activity was probably due to the cytotoxicity of the test item.
Under the condition of this study the test item is therefore considered as non-sensitiser.

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: Overall Induction

Overall Induction	Conc. [µM]	Relative Fold Induction^1)				Sign.	Experiment 3
		Exp. 1	Exp. 2	Mean	SD		Conc. [µM]	Relative Fold Induction^1)	Sign.
Solvent Control	-	1.00	1.00	1.00	0.00		-	1.00
Positive Control	4.00	1.23	1.38	1.31	0.11		4.00	1.13
	8.00	1.20	1.19	1.19	0.01		8.00	1.10
	16.00	1.55	1.30	1.42	0.18		16.00	1.29
	32.00	2.13	2.57	2.35	0.31	*	32.00	1.68	*
	64.00	4.34	4.64	4.49	0.22	*	64.00	2.36	*
Test Item	0.98	1.20	1.29	1.24	0.07		0.64	[1.57]	*
	1.95	1.14	1.11	1.12	0.02		0.85	1.35
	3.91	1.25	0.82	1.03	0.30		1.13	1.27
	7.81	2.29	1.57	1.93	0.51		1.50	1.17
	15.63	1.57	1.62	1.60	0.03	*	2.01	1.31
	31.25	0.02	0.01	0.02	0.01		2.67	1.17
	62.50	0.00	0.00	0.00	0.00		3.56	1.32
	125.00	0.00	0.00	0.00	0.00		4.75	1.13
	250.00	0.00	0.00	0.00	0.00		6.33	1.22
	500.00	0.00	0.00	0.00	0.00		8.44	1.32
	1000.00	0.01	0.00	0.00	0.00		11.25	1.78	*
	2000.00	0.00	0.00	0.00	0.00		15.00	1.77	*

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

[outlier] This value was regarded as biological outlier and was not included in further evaluations, as no dose-effect for luciferase activity was observed.

Table 2: Overall calculated values

Parameter	Experiment 1	Experiment 2	Experiment 3	Mean	SD
EC1.5[µM]	4.86	7.47	9.55	7.29	2.35
Imax	2.29	1.62	1.78	1.90	0.35
IC30[µM]	10.82	10.18	11.82	10.94	0.83
IC50[µM]	13.16	12.01	n.a.	12.59	0.81

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item 5’-O-[bis(4-methoxyphenyl)phenylmethyl]-2’-deoxythymidine, 3’-[2-cyanoethyl N,N-bis(1-methylethyl)phosphoramidite] 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 skin sensitisation study conducted according to OECD 442D with 5’-O-[bis(4-methoxyphenyl)phenylmethyl]-2’-deoxythymidine, 3’-[2-cyanoethyl N,N-bis(1-methylethyl)phosphoramidite] (99.7% purity), the sensitisation potential of the test item was assessed keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™.Cells were incubated with the test item for 48 h at 37 °C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement. Within all three experiments the induced the gene activity was very close to the cytotoxic levels. However, no dose-response relationship for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. It is assumed that the induction of luciferase activity was probably due to the cytotoxicity of the test item. Based on these results, the test item is not considered to be a skin sensitizer under the UN GHS criteria.

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019-10-23 to 2020-01-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

adopted 22 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was placed into an appropriate container on a tared balance and DMF was added (weight per weight). The different test item concentrations were prepared individually. The preparations were made freshly before each dosing occasion.

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc, Postbus 6174, 5960 AD Horst, The Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8-9 weeks (pre-test), 8 weeks (main study)
- Weight at study initiation: 19.5-20.8 (pre-test), 16.5-20.7 g (main study)
- Housing: Animals were housed in a group, Makrolon Type II (pre-test) / III (main study) cages, with wire mesh top
- Diet: ad libitum, 2018C Teklad Global 18% protein rodent diet (certified)
- Water: ad libitum, tap water
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 (20-24)
- Humidity (%): approx. 45-65
- Photoperiod (hrs dark / hrs light): 12/12, artificial light (6.00 a.m.-6.00 p.m.)

Vehicle:

dimethylformamide

Concentration:

5%, 10%, 25%, 50% (w/w)

No. of animals per dose:

- Pre-test: 2 female mice/group
- Main experiment: 5 female mice/group

Details on study design:

See "Any other information on materials and methods incl. tables" below.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The mean values and standard deviations were calculated in the body weight tables. Where appropriate, the EC3 value were calculated according to the equation EC3 = (a-c) [(3-d)/(b-d)] + c, where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot. All calculations conducted on the DPM values and the ear weights, were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.
Within the program a statistical analysis conducted on the DPM values and the ear weights in order to assess whether the difference was statistically significant between the test item groups and negative control group. Statistical significance was set at the five per cent level (p< 0.05). Additionally, for the DPM values, the Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers. No outliers were detected in both statistical tests. However, both biological and statistical significance will be considered together.

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:

1.8

Test group / Remarks:

5%

Key result

Parameter:

SI

Remarks:

mean of five animals

Value:

1.4

Test group / Remarks:

10%

Key result

Parameter:

SI

Remarks:

mean of five animals

Value:

2

Test group / Remarks:

25%

Key result

Parameter:

SI

Remarks:

mean of five animals

Value:

1.8

Test group / Remarks:

50%

Cellular proliferation data / Observations:

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

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

CLINICAL OBSERVATIONS:
- No deaths occured during the study period. No signs of systemic toxicity were observed during the study period. From day 1 to 6, the animals showed an erythema of the ear skin (Score 1 to 2). Additionally, scaly ears and fur loss was observed.

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 an erythema of the ear skin (Score 1 to 2). Additionally, fur loss, stiffened ears, scaly ears and erythema of scalp were observed in the animal treated with the high dose of the test item. In the animal treated with the low dose, substance residuals and scaly ears were observed. A maximum increase in ear thickness of 10.4% (animal 1) and 7.5 % (animal 2) was observed. No excessive increase in ear weights was observed in both animals.

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

						Group calculation
Test item (DMT-dT-Phosphoramidite) 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	13	-	-	-	-	-
-	-	background 2	21	-	-	-	-	-
Vehicle Control Group (DMF)	1	1	1457	1440	-	2378.0	709.0	1.0
	1	2	2352	2335	-
	1	3	2588	2571	-
	1	4	3414	3397	-
	1	5	2164	2147	-
5% DMT-dT- Phosphoramidite	2	6	3310	3293	1.4	4323.0	1598.8	1.8
	2	7	2841	2824	1.2
	2	8	3492	3475	1.5
	2	9	6526	6509	2.7
	2	10	5531	5514	2.3
10% DMT-dT- Phosphoramidite	3	11	1863	1846	0.8	3287.8	1085.6	1.4
	3	12	3225	3208	1.3
	3	13	2798	2781	1.2
	3	14	4716	4699	2.0
	3	15	3922	3905	1.6
25% DMT-dT- Phosphoramidite	4	16	2049	2032	0.9	4849.0	1867.8	2.0
	4	17	4839	4822	2.0
	4	18	4394	4377	1.8
	4	19	6138	6121	2.6
	4	20	6910	6893	2.9
50% DMT-dT- Phosphoramidite	5	21	2528	2511	1.1	4270.4	1302.9	1.8
	5	22	5766	5749	2.4
	5	23	3460	3443	1.4
	5	24	5165	5148	2.2
	5	25	4518	4501	1.9

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, in a mouse local lymph node assay, the test item DMT-dT-Phosphoramidite 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-Phosphoramidite in DMF at concentrations of 5%, 10% and 25% and 50% (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. From day 1 to 6, the animals showed an erythema of the ear skin (Score 1 to 2). Additionally, scaly ears and fur loss was observed.

No mortality or effects on body weight occurred during the study. None of the four tested concentrations exceeded a stimulation index of 3 (1.8 (5%), 1.4 (10%) and 2.0 (25%) and 1.8 (50%)). As a consequence, an EC3 value could not be calculated. In this study, DMT-dT-Phosphoramidite is not a dermal sensitizer.

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: N#CCCOP(OC4CC(OC4(COC(c1ccccc1)(c2ccc(OC)cc2)c3ccc(OC)cc3))N5C=C(C(=O)NC5(=O))C)N(C(C)C)C(C)C

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 = 0.644
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 = 0.492
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: 3 descriptors 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 (7 unknown fragments and 4 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: N#CCCOP(OC4CC(OC4(COC(c1ccccc1)(c2ccc(OC)cc2)c3ccc(OC)cc3))N5C=C(C(=O)NC5(=O))C)N(C(C)C)C(C)C

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 = 0.24
Explanation: the predicted compound is outside the Applicability Domain of the model.

Similar molecules with known experimental value
Similarity index = 0.68
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 = 0.537
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 = 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 (5 unknown fragments and 1 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 = 0.644), and similar molecules found in the training set had experimental values that disagree with the predicted value, and the descriptors 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 = 0.68) and similar molecules were found in the training set which had experimental values that disagree 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.

Reference 5

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-07-05 to 2018-11-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

adopted 04 February 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

In order to replace in vivo experiments validation studies on alternative, mechanistically based in chemico and in vitro test methods on skin sensitisation were conducted under the auspices of European Centre for the Validation of Alternative Methods (ECVAM) and have been considered scientifically valid for the evaluation of the skin sensitisation hazard of chemicals. It was concluded that the direct peptide reactivity assay (DPRA) showed evidence of being a reliable and relevant method to test for skin sensitisation testing. However, only combinations of several non-animal testing methods within an Integrated Approach to Testing and Assessment (IATA) will be able to fully substitute for the animal test currently in use.

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was pre-weighed in a glass vial and was dissolved in methanol. A solution with a concentration of 100 mM was prepared.

Details on the study design:

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

Controls: Reference controls, co-elution controls and a positive control (PC) were set up in parallel to the test item in order to confirm the validity of the test.
Positive Control:
Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was dissolved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.
Co-elution controls:
were set up in parallel to sample preparation but without the respective peptide solutions. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides.
Reference Control:
Reference Controls (RCs) were set up in parallel to sample preparation in order to verify the validity of the test run.
Reference control A was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Three replicates of this RC were injected in the beginning of each HPLC run.
Reference control B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Three replicates of this RC were injected in the beginning and in the end of each HPLC run.
Two reference controls C were set up for the test item and for the positive control. RC C for the positive control was prepared using acetonitrile. RC C for the test item was prepared using the respective solvent (methanol) used to solubilise the test item. The controls were used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally, reference control C was used to calculate PPD.
The RC Cs with acetonitrile and the test item solvent (methanol) were included in every assay run for both peptides and were injected just before the positive control and the test item samples.

Pre-Experiments
Solubility of the test item was determined prior to the main experiment and was tested at the highest final concentration applied in the study (100 mM). Solubility was investigated with the following solvents suitable for the test:
- acetonitrile
- dist. water
- dist. water:acetonitrile 1:1 (v/v)
- isopropanol
- methanol
- 1,4-butanediol
- N,N-dimethylformamide
- Ethanol
- tert. Butanol
The test item was not soluble in dist. water, dist. water:acetonitrile 1:1 (v/v), isopropanol, 1,4-butanediol and tert. Butanol. The test item was completely soluble in acetonitrile, methanol, N,N-dimethylformamide and ethanol. Therefore, acetonitrile was chosen as suitable vehicle for the main experiments.

Experimental Procedure
Incubation of the Test Item with the Cysteine and Lysine Peptides
The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 for the cysteine peptide, 1:50 for the lysine peptide). The reaction solutions were incubated in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. Reference controls, co-elution controls as well as the positive control were set up in parallel. Samples were prepared according to the scheme described in Table 1 in box "Any other information on materials and methods". Test item solutions were inspected on a visual basis for the formation of precipitates, turbidity and phase separation prior and after HPLC analysis. If a precipitate or phase separation was observed after the reaction period and prior to the HPLC analysis, samples might have been centrifuged at low speed (100 - 400 x g) to force precipitates to the bottom of the vialAfter the incubation period of 24 ± 2 h the test item was analysed in triplicate for both peptides using the following HPLC procedure.

Preparation of the HPLC Standard Calibration Curve
A standard calibration curve was generated for each peptide. Peptide standards were prepared in a solution of 20% acetonitrile: 80% buffer (v/v) using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide (dilution buffer (DB)). A serial dilution of the peptide stock solution (0.667 mM) using the respective DB was performed, resulting in 7 calibration solutions for each peptide (0, 0.017, 0.033, 0.067, 0.134, 0.267 and 0.534 mM).

HPLC Preparation and Analysis
Peptide depletion was monitored by HPLC coupled with an UV detector at λ = 220 nm using a reversed-phase HPLC column (Zorbax SB-C-18 2.1 mm x 100 mm x 3.5 micron) as preferred column. The entire system was equilibrated at 30 °C with 50% phase A and 50% phase B for at least 2 hours before running the analysis sequence. The HPLC analysis was performed using a flow rate of 0.35 mL/min and a linear gradient from 10% to 25% acetonitrile over 10 minutes followed by a rapid increase to 90% acetonitrile. The column was re-equilibrated under initial conditions for 7 minutes between injections. Equal volumes of each standard, sample and control were injected.
The analysis was timed to assure that the injection of the first sample started 22 to 26 hours after the test chemical was mixed with the peptide solution. The HPLC run sequence was set up in order to keep the HPLC analysis time less than 30 hours.

Data Analysis
The concentrations of the cysteine and lysine peptide were determined in each sample from absorbance at λ = 220 nm, measuring the area of the appropriated peaks (peak area (PA)) and calculating the concentration of peptide using the linear calibration curves derived from the standard solutions. The percent peptide depletion (PPD) was calculated according to the following formula:
PPD = [1 – (Peptide PA in the replicate injection/mean peptide PA in reference control C)] x 100
The absorbance at λ = 258 nm was also monitored for the samples of the test item and the reference controls as a co-elution control. The ratio of the peak areas (220 nm / 258 nm) was checked for consistency between reference control and test item samples. If this ratio was not consistent, a co-elution was assumed and the evaluation would be adjusted accordingly.
Sensitising potential of the test item is predicted from the mean cysteine and lysine PPD value. The test item is considered positive and to be a skin sensitiser if the mean depletion of both peptides exceeds the threshold of prediction model 1 (see Table 2 in box "Any other information on materials and methods"). Negative depletion is considered as “0” when calculating the mean.
By using the prediction model 1 (cysteine 1:10 / lysine 1:50 prediction model), the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers. Application of the prediction model for assigning a test item to a reactivity class (i.e. low, moderate or high reactivity) may perhaps prove useful to inform potency assessment within the framework of an IATA. In the framework of an IATA the test substance may be considered as non-sensitiser to skin if the mean depletion of both peptides is below 6.38%.

In case of co-elution of the test item with a peptide peak, the peak cannot be integrated correctly and the calculation of the PPD is not possible. If severe co-elution occurs with both peptides then the analysis was reported as "inconclusive". In cases where the co-elution occurs only with the lysine peptide, prediction model 2 can be applied (cysteine 1:10 prediction model) (see Table 3 in box "Any other information on materials and methods").

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 65.80% (prediction model 1) and 71.52% (prediction model 2).

Key result

Parameter:

other: Mean percent of peptide depletion

Value:

0

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Due to the observed precipitation after the incubation period in the cysteine and the lysine peptide samples containing also test item, no prediction can be made.

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: yes

For individual results see Tables 4 to 6 in box "Any other information on results incl. tables".

Table 4:   Depletion of the Cysteine Peptide

Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	4.6010	0.1397	71.75	71.52	0.28	0.39
	4.6220	0.1404	71.62
	4.6890	0.1424	71.21
Test Item	16.8030	0.5090	0.00	0.00	0.00	n.a.
	16.6760	0.5052	0.00
	16.6020	0.5029	0.00

 

Table 5:   Depletion of the Lysine Peptide

Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	5.4360	0.2012	60.55	60.08	1.12	1.86
	5.3900	0.1995	60.88
	5.6770	0.2101	58.80
Test Item	14.1170	0.5236	0.00	0.00	0.00	n.a.
	13.9410	0.5170	0.00
	13.9640	0.5179	0.00

 

Table 6: Prediction of sensitising potential of the test item

	Prediction Model (Cysteine 1:10 and Lysine 1:50)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	0.00	Minimal Reactivity	negative
Positive Control	65.80	High Reactivity	positive

Interpretation of results:

study cannot be used for classification

Conclusions:

In this study (performed in accordance with OECD guideline 442C) under the given conditions DMT-dT-Phosphoramidite showed minimal reactivity (as indicated by peptide depletion) towards both peptides. However, due to the observed precipitation and phase separation, the prediction model does not apply and a prediction cannot be made.

Executive summary:

In the in chemico sensitisation study performed in accordance with OECD guideline 442C, the test item DMT-dT-Phosphoramidite was dissolved in acetonitrile. Based on its molecular weight of 744.81 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by high-performance liquid chromatography (HPLC). For the 100 mM solution of the test item, precipitation was observed when diluted with the peptide solutions.

The stock solution of the test item showed no reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 6.38% (0.00%). Since precipitation was observed in the cysteine peptide and phase separation was observed in the lysine peptide, a test item concentration of 100 mM as well as the full contact of peptide and test item is not guaranteed. According to the evaluation criteria in the guideline, no firm conclusion on the lack of reactivity should be drawn from a negative result, if a test chemical is tested in concentration < 100 mM. Therefore, no prediction can be made.

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.