Registration Dossier
Registration Dossier
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EC number: 255-003-1 | CAS number: 40615-39-2
Endpoint summary
Administrative data
Description of key information
The skin sensitisation potential and potency of the substance 5’-O-(4,4’-dimethoxytrityl)thymidine was assessed in three skin sensitisation assays (OECD 442D, OECD 442E and GardTM assay), one in chemico assay (OECD 442C) and in one in vivo LLNA study (OECD 429).
In the first study, conducted according to OECD 442E with the substance in DMSO, 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 a 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 a third study, protein depletion was analysed in accordance with OECD guideline 442C. Due to low solubility of the test item it was not possible to conduct the OECD 442C assay. Due to the observed precipitation, the prediction model does not apply, and a prediction could not be made.
In a fourth study the test item was tested in the GARD^TM assay. Based on the results from the GARDskin biomarker set, the test item can be considered to be a sensitiser. In addition, based on the results from the GARDpotency genomic biomarker set, sub-categorisation into Cat 1A is applicable.
In addition to the contradictory in vitro/in chemico data, suitable information from an in vivo study conducted according to OECD 429 was used to assess the skin sensitising potential of the target substance. 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
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2017-12-05 to 2018-04-26
- 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 04 February 2015
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- activation of keratinocytes
- Justification for non-LLNA method:
- The induction of the Keap1-Nrf2-ARE signalling pathway by small electrophilic substances such as skin sensitisers was reported by several studies and represents the second key event of the skin sensitisation process as described by the AOP. Therefore, the KeratinoSens™ assay is considered relevant for the assessment of the skin sensitisation potential of chemicals.
- Specific details on test material used for the study:
- TREATMENT OF TEST MATERIAL PRIOR TO TESTING
All test item solutions were freshly prepared immediately prior to use. The test item was dissolved in dimethyl sulfoxide. A stock solution of 200 mM was prepared by pre-weighing the test material into a glass vial. Based on the DMSO stock solution, serial dilutions were made using the solvent to obtain twelve 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 resulting in a 4% share of the solvent. These 1:25 diluted test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent 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 11, experiment 1; passage 6, experiment 2) were used. Cells were cultured in 75 cm² culture flasks (Greiner) in maintenance medium at 37 +/- 1 °C and 5% CO2 in a humidified incubator. For test material exposure, cells were cultured in medium.
LUCIFERASE ASSAY SYSTEM:
The luciferase activity was determined using the following products purchased from Promega. All components were used according to the instructions of the manufacture manual. The kit (Promega, Cat. No.: E1501, Lot No.: 0000276369) consisted of the following components relevant for this study:
- 10 vials Luciferase Assay Substrate (lyophilized)
- 10 x 10 mL Luciferase Assay Buffer
If freshly prepared, Luciferase Assay Substrate was dissolved in Luciferase Assay Buffer. If thawed from -80 °C, Luciferase Assay Reagent was allowed to equilibrate to room temperature prior to use.
Luciferase Cell Culture Lysis 5x Reagent
The kit (Promega, Cat. No.: E1531, Lot No.: 0000245622) consisted of the following components relevant for this study:
- 30 mL Luciferase Cell Culture Lysis 5x Reagent
Prior to use lysis buffer was diluted 1:5 with dist. water (Sigma; Lot No.: RNBG3519)
DOSE GROUPS:
Negative Control: DMSO: 1% (v/v) in test item exposure medium
Positive Control: 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:
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 shortly before prepared 25 times diluted master concentrations were transferred the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item.
All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated 96-well plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.
LUCIFERASE ACTIVITY:
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS (Gibco Life Science; Lot No.: 1909266, 1877596). Subsequently 20 µL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light.
Plates with the cell lysate were placed in the plate reader (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 over the weekend (experiment 1 and 2). After the incubation period the plate was shaken for 10 min and the optical density (OD) was measured at λ = 600 nm.
DATA ANALYSIS:
For every concentration showing >1.5 fold luciferase activity induction, statistical significance (p <0.05) was calculated using a two-tailed Student’s t-test comparing the luminescence values for the three replicated samples with the luminescence values in the solvent (negative) control wells. The lowest concentration with >1.5 fold luciferase activity induction was the value determining the EC1.5 value. It was checked in each case whether this value was below the IC30 value, indicating that there was less than 30% reduction on cellular viability at the EC1.5 determining concentration.
PREDICTION MODEL:
The test item is considered positive in accordance with UN GHS “Category 1” for skin sensitisation if the following conditions were met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p< 0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is <1000 µM
- an apparent overall dose-response for luciferase induction
If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <1000 µM is considered as inconclusive. 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 (3.29 in experiment 1; 2.72 in experiment 2).
- The calculated EC1.5 was between 7 and 34 µM (11.09 µM in experiment 1; 23.97 µM in experiment 2). - Run / experiment:
- other: Mean of Experiment 1 and 2
- Parameter:
- other: max luciferase activity (lmax) induction
- Value:
- 1.38
- 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 >1.5-fold in tested concentration range; no EC1.5 value could be calculated
- Key result
- Run / experiment:
- other: Experiment 1, at 3.91 µM
- Parameter:
- other: max luciferase activity (Imax) induction
- Value:
- 1.48
- 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 >1.5-fold in tested concentration range; no EC1.5 value could be calculated
- Key result
- Run / experiment:
- other: Experiment 2, at 3.91 µM
- Parameter:
- other: max luciferase activity (Imax) induction
- Value:
- 1.29
- 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 >1.5-fold in tested concentration range; 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:
- Acceptance criteria: The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition.
- Acceptance criteria met for positive control: Yes
For individual results see Table 1 in box 'Any other information on results incl. tables'. - Interpretation of results:
- GHS criteria not met
- Remarks:
- Test item is considered a non-sensitiser
- Conclusions:
- In this study under the given conditions the test item 5’-O-(4,4’-Dimethoxytrityl)thymidine did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, 5’-O-(4,4’-Dimethoxytrityl)thymidine can be considered to be a non-sensitiser.
- Executive summary:
In a dermal sensitisation study conducted according to OECD 442D with 5’-O-(4,4’-Dimethoxytrityl)thymidine (purity 100%) in DMSO, the sensitisation potential of the test item was assessed on the basis of the activation of keratinocytes using the in vitro KeratinoSens™. Cells were incubated with the test item for 48 h at 37 °C and later checked for luciferase activity.
Sensitisation was scored by measuring maximum luciferase activity induction (Imax), cell viability and EC1.5. For both Experiment 1 and 2, Imax was less than a 1.5 fold increase and the EC1.5 could not be calculated.
Therefore, in this study, the test item 5'-O-(4,4'-Dimethoxytrityl)thymidine is considered to be a skin non-sensitiser.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2017-12-05 to 2018-05-03
- 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 immunological relevant cell surface markers with the skin sensitising potential of a chemical has been reported and represents the third key event in the skin sensitisation process as described by the AOP. This method that measures the markers of DC activation, based on DC-like cell line THP-1 is considered relevant for the assessment of the skin sensitisation potential of chemicals. Moreover, this test method is able to detect chemicals that cause skin sensitisation and allows hazard identification.
- 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 dimethyl sulfoxide (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. Precipitates were observed when diluted 1:250 in cell culture medium in the dose finding experiment 1 and 2. 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 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 10^6 cells/mL.
Cells were cultured in 75 cm^2 culture flasks (Greiner) in Roswell Park Memorial Institute medium (RPMI-1640) 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 positive controls. For the reactivity check of the cell batch additional negative and positive controls were included. DNCB at a final concentration of 4 µg/mL and nickel sulphate at a final concentration of 100 µg/mL served as positive control while lactic acid 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.
EXPERIMENTAL PROCEDURE:
DOSE FINDING STUDY:
Starting from 500 mg/mL (experiment 1) and 125 mg/mL (experiment 2) 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 into a 24 well flat-bottom plate (1 x 10^6 cells/well).
The solvent controls, 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 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 10,000 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. 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; CV75/1.22; CV75/1.23; CV75/1.24; CV75/1.25; CV75/1.26. 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 controls, 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, anti-CD54, or mouse IgG1 (isotype) antibodies in the dark for 30 min. 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 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. - Positive control results:
- The positive controls DNCB and NiSO4 led to upregulation of the cell surface markers CD54 and CD86 in both experiments. The threshold of 150% for CD86 (334% experiment 1; 347% experiment 2) and 200% for CD54 (353% experiment 1; 246% experiment 2) were clearly exceeded.
- Run / experiment:
- other: 1
- Parameter:
- other: CD86 upregulation
- Remarks:
- at highest tested concentration of 16.32 µg/mL
- Value:
- 214
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Run / experiment:
- other: 1
- Parameter:
- other: CD54 upregulation
- Remarks:
- at highest tested concentration of 16.32 µg/mL
- Value:
- 991
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Run / experiment:
- other: 2
- Parameter:
- other: CD86 upregulation
- Remarks:
- at highest tested concentration of 16.32 µg/mL
- Value:
- 314
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Run / experiment:
- other: 2
- Parameter:
- other: CD54
- Remarks:
- at highest tested concentration of 16.32 µg/mL
- Value:
- 977
- 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 - Interpretation of results:
- other: the test item is considered to be a skin sensitizer
- Conclusions:
- In this study under the given conditions the test item 5’-O-(4,4’-Dimethoxytrityl)thymidine did upregulate the cell surface markers in two independent experiment runs. Therefore, the test item can be considered to positive in this test.
- Executive summary:
In a skin sensitisation study conducted according to OECD 442E with 5’-O-(4,4’-Dimethoxytrityl)thymidine (Purity 100%) in DMSO, 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 upregulated above the threshold of 200% in both experiments. Since at least one of the cell surface markers clearly exceeded the threshold in at least two independent experiments 5’-O-(4,4’-Dimethoxytrityl)thymidine can be considered to be a skin sensitiser.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2019-01-07 to 2019-07-03
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- The study is conducted according to the internal requirements from Senza Gen AB (DB-Alm Protocol GARD – Genomic Allergen Rapid Detection (GARDskin)). The Genomic Allergen Rapid Detection (GARD^TM) is an in vitro assay designed to predict the ability of chemical substances to induce skin sensitisation based on the analysis of the relative expression levels of a biomarker signature of 196 genes using a human myeloid leukemia cell line called SenzaCell. The GARD^TM is based on chemical stimulation of the SenzaCell line, acting as an in vitro model of human Dendritic Cells (DCs). The readout of the assay is a transcriptional quantification of the genomic predictors, collectively termed the GARD^TM Prediction Signature (GPS), using Nanostring nCounter technology.
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- activation of dendritic cells
- Justification for non-LLNA method:
- The GARD^TM method mimics the immune system. It predicts the ability of chemical compounds to induce skin sensitisation. Many in vitro tests for sensitisers on the market are fairly simple to perform, but they are often not enough to give reassuring results. Genomics-based models offer reliable alternatives to animal testing. Since GARD^TM is examining the expression from more than a hundred genes that are all collectively relevant for the AOP of skin sensitisation, several key events are addressed in one single test.
- Specific details on test material used for the study:
- TREATMENT OF TEST MATERIAL PRIOR TO TESTING
All test item solutions were freshly prepared immediately prior to use. The test item was dissolved in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity ≥ 99%). Vortex mixing was used to aid solubilisation. After a suitable solvent was determined, the solubility of the test item solution was tested after a 1:100 dilution in semi-complete media. The solvent was present at a constant volume ratio of 0.1% (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control. A correction factor of 1.022 was applied to correct for the purity of the test item. - Details on the study design:
- Skin sensitisation (In vitro test system)
- Details on study design:
CELL LINE
The test was carried out using human myeloid leukemia cell line Senza cells provided by SenzaGen AB. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and sub-cultured at least 2 weeks before they were used in the in vitro GARD^TM. Only cells at a low passage number (< 16) were used. Cells are routinely passaged every 3-4 days at a density of 0.2 x 10^6 cells/mL. Cells are cultured in 175 cm² culture flasks (Greiner) in MEM/Alpha Modification with L-glut, Ribo- Deoxyribo (Thermo Scientific; Cat. No.: SH30265.01) supplemented with 20% fetal bovine serum (FBS, Gibco Life Science, Cat No.: 10270-106) (= semi-complete medium), and freshly added 40 ng/mL GM-CSF (Miltenyi Biotec, Cat No.: 130-093-868) in a humidified incubator at 37 +/- 1 °C and 5% CO2 (complete medium).
DOSE GROUPS
- Medium Control: semi-complete medium
- Solvent Control: 0.1% (v/v) for DMSO or a different solvent for the test item and the positive control
- Positive Control: 75 µM PPD (p-Phenylenediamine)
- Test Item: Input finder: 9 concentrations of the test item down to 1 mM stock solution; Main stimulation: 1 concentration, which leads to a relative cell viability of 90 ± 5%.
PRE-EXPERIMENTS:
Solvent Finding:
Solubility of the test item was determined prior to the Input Finder assay. The test item was dissolved in 100% DMSO at a final concentration of 500 mM. If soluble, DMSO was used as the solvent. If the test item was not soluble in DMSO at 500 mM, the test item was dissolved at 500 mM in sterile water. If soluble, sterile water was used as the solvent. If the test item was not soluble in sterile water at 500 mM the highest soluble concentration was determined by diluting the solution from 500 mM to a minimal concentration of 1 mM in both solvents. Heating up to 37 °C and vortexing was applied to achieve complete solubility. If a solvent was determined, the solubility of the test item solution in semi-complete medium was tested by a 1:100 dilution. If precipitation, phase separation of turbidity is observed the test item was further diluted in the solvent and tested again in semi-complete medium at lower concentrations after a 1:100 dilution. This was done until no precipitation, phase separation of turbidity was observed in semi-complete medium.
Phenotypic Quality Control:
At the same day, as the chemical stimulation was performed, the cells was quality controlled by a phenotypic analysis. 2 x 10^5 cells were transferred six times into FACS tubes. The cells were washed twice with Dulbecco’s phosphate buffered saline (DPBS) containing 0.5-1% bovine serum albumin (BSA; i.e. FACS buffer). After washing the cells were resuspended in 50 µL FACS buffer and stained with the surface markers (e.g. CD86, CD54, CD34, CD10) for 15 minutes at 2-8 °C in the dark. After staining the cells were washed once with 1 mL FACS Buffer and the cell pellets were resuspended in 200 µL FACS buffer for analysis. If the phenotypic quality control passes all criteria, the cells were used for stimulation.
EXPERIMENTAL PROCEDURE
Input Finder Assay:
Starting from the highest soluble concentration in the most applicable solvent and medium determined in the solvent finding experiment, maximal nine stock solutions (i.e. nine concentrations) down to 1 mM were prepared. (i.e. 500 mM – 400 mM – 300 mM – 200 mM – 100 mM – 50 mM – 10 mM – 5 mM – 1 mM). These stock solutions were further diluted 100-fold into semi-complete medium giving the stock B solutions. For testing, the highest concentration was 500 µM, even if this concentration is non-toxic. The stock B solutions were finally used for treatment by adding the test item to the SenzaCell suspension in complete medium in a 24-well plate to achieve a further 10-fold dilution. For testing, SenzaCells were pre-cultured for at least 72 h - 96 h in culture flasks at a cell density of 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 semi-complete medium at a density of 2.22 x 10^5 cells/mL. Then 1.8 mL of cell suspension was seeded into a 24-well plate-bottom plate (2 x 10^5 cells/well).
The stock B solutions of the solvent control and the dilution range of the test item was mixed 1:10 (v/v) with the cell suspension prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% ± 0.5% CO2.
After 24 h ± 0.5 h of exposure, cells were transferred and split into two FACS tubes. FACS buffer was added and the cells were collected by centrifugation (approx. 300x g). The supernatant was discarded and the remaining cells were washed once with FACS buffer. After washing, cells were re-suspended in 50 µL propidium iodide (PI) solution (1 µg/mL) in FACS buffer. Cells were stained ~ 10 minutes at 2-8 °C in the dark. After staining cells were washed with FACS buffer again and re-suspended in 200 µL FACS buffer.
The PI uptake of the cells (as an indicator of 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 10.000 living (PI negative) cells were acquired and cell viability was calculated for each test concentration. Test items that induced cytotoxicity was used for main stimulation at the concentration that induces 90% ± 5% relative viability (RV90). Test items that induced no or week cytotoxicity (≥ 95.5% relative viability) were tested at 500 µM final concentration or at the highest soluble concentration. If cytotoxic effects were determined, but the relative viability is not between the range of 90% ± 5% the input finder was repeated with another dilution range. The determined concentration had to be 1000-fold higher for the stock solution for the main stimulation experiment (= GARD input concentration).
Main Stimulation:
A stock solution of the positive control (75 mM) in DMSO and a stock solution 1000-fold higher as the GARD input concentration of the test item in the solvent determined in the solvent finding pre-experiment were freshly prepared immediately before use.
The stock solution of the positive control, pure DMSO as a negative control, the stock solution of the test item and the solvent of the test item were diluted 100-fold into semi-complete medium (= stock B) to achieve the in-well concentration of 0.1% DMSO.
For testing, SenzaCells were pre-cultured for at least 72 h - 96 h in culture flasks at a cell density of 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 semi-complete medium at a density of 2.22 x 10^5 cell/mL. Then 3.6 mL of cell suspension were seeded into a 12 well plate-bottom plate (2 x 10^5 cells/well). The stock B solutions of the positive control, the solvent control of the test item input concentration and the solvent of the test item were mixed 1:10 (v/v) with the cell suspension prepared in the 12-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% ± 0.5% CO2. The cells were stained to check the quality of the cells. After 24 h ± 0.5 h of exposure, 1 mL of the cells were transferred three times into small reaction tubes and further 500 µL were transferred twice into two FACS tubes. The cells of the reaction tubes were collected by centrifugation (approx. 300 x g), re-suspended in 500 µL TRIzol and frozen at < -20 °C. The cells of the FACS tubes were washed twice with FACS buffer. After washing, cells were re-suspended in 50 µL propidium iodide (PI) solution (1 µg/mL) in FACS buffer. Cells were stained for approx. 10 minutes at 2-8 °C in the dark. After staining, cells were washed with FACS buffer again and will be re-suspended in 200 µL FACS buffer.
The PI uptake of the cells (as an indicator of 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 10.000 living (PI negative) cells were acquired and cell viability will be calculated for each sample. If the cell viability passes the criteria the RNA of the TRIzol samples were collected.
RNA Isolation:
The first tube of each TRIzol sample triplicate was thawed slowly on ice. The samples were centrifuged to remove particulate debris and the supernatant was transferred into new tubes. An equal volume of 95% - 100% ethanol was added and the whole mixture was transferred on the column of a Zymo-SpinTM IIC Columns (ZymoResearch Cat. R5052).
The flow through was discarded and the column was washed twice with 400 µL with the Direct-zolTM RNA PreWash. The flow through was discarded again. After the pre-washing step the column was washed with 700 µL RNA wash buffer. The flow through was discarded again and the column was placed in a new RNAse-free tube. The RNA was eluted with 25 µL RNAse-free water. After centrifugation the collected RNA was transferred on the column again to elute the whole RNA. The RNA was stored at -80 °C for shipment.
ENDPOINT MEASUREMENT
RNA Quality Control:
The RNA quality and quantity were measured with an Agilent 2100 BioAnalyser. If the RNA sample did not pass the quality control criteria the RNA had to be isolated from the last two TRIzol samples and quality had to be measured again. RNA samples which had passed the criteria undergo Nanostring endpoint measurement with an appropriate code set for GARDskin.
Nanostring Hybridisation:
All hybridization samples used a total RNA concentration of 100 ng. All RNA samples were diluted to 20 ng/µL with RNA-free water to use 5 µL of each for hybridization. For hybridization mix 8 µL of the master mix (provided in the Nanostring master kit), 5 µL RNA sample (20 ng/µL) and 2 µL of the capture code set were mixed. The whole approach was incubated in a thermocycler at 65 °C for 24 h ± 0.5 h.
Setting Up the Nanostring Preparation and Measurement:
The nCounter Prepstation (Nanostring) was prepared as described in the manual. After hybridization the samples were removed from the thermocycler and spinned down before opening. The samples were placed in the nCounter Prepstation, too. The preparation of the nanostring cartridges took around 3 hours. The cartridges were analysed by an nCounter Digital Analyser (Nanostring).
DATA ANALYSIS
Calculation of the relative viability:
The cell viability was measured with FACS. The FACS data analysis was performed using the software BD FACS DIVA 6.0. Further data analysis like calculation of the GARD input concentration and calculation of the relative viability was performed using the software Microsoft Excel 2010. The relative viability percentage calculation is based on the following equation: Rv= (Vs/Vc)*100
Nanostring Data Analysis:
The NanoString raw data files were analysed using the GARD Data Analysis Application provided from SenzaGen AB. If the Decision Value (DV) showed sensitisation potential, the RNA samples could be measured with a different code set again to get information on the potency of the substance to make categorization into 1A and 1B.
ACCEPTANCE CRITERIA
Criteria for the phenotypic quality control:
Phenotypic biomarker: Acceptance range for positive cells (%)
- CD86: 10-40
- CD54: +
- HLA-DR: +
- CD80: < 10
- CD34: +
- CD14: +
- CD1α: +
Phenotypic biomarker: Acceptance range for negative cells (%)
- Absolute viability: ≥ 84.5
A stimulation of the cells can be done if the criteria are fulfilled.
Criteria of the relative viability quality control:
The cell viability of the cell main stimulation experiment has to fulfil the following criteria:
- Substances that are expected to induce cytotoxicity have a relative viability of 84.5% - 95.4%
- Substances that are not expected to induce cytotoxicity have a relative viability of ≥ 95.5%
- The unstimulated control should have an absolute viability of ≥ 84.5%
If the criteria for the relative viability of the test item are not fulfilled because of an incorrect GARD input concentration, the test item has to be retested to find the correct GARD input concentration.
If one of the controls does not fit the criteria because of a technical error, the control has to be treated twice in the next experiment. If it happens in the third main stimulation a fourth main stimulation has to be done.
If the unstimulated cells do not reach the acceptance criteria, a re-run has to be done.
Criteria for the RNA quality control:
The RNA samples have to fulfil the following criteria:
- The RNA concentration of a qualified sample should be ≥ 20 ng/µL
- The RNA integrity number (RIN) for a qualified sample should be ≥ 8.0
PREDICTION
The prediction is defined as described below:
- If the mean decision value (DV) of biological replicate samples is ≥ 0, the substance is classified as a sensitiser
- If the mean decision value (DV) of biological replicate samples is < 0, the substance is classified as a non-sensitiser. - Positive control results:
- The positive control sampes induced the appropriate response. The decision values of the positive control are > 0 (10.17; 9.37; 10.91) in the GARDskin analysis. In the GARDpotency analysis the decision values of the positive control were > 0 ((8.36, 8.69, 8.72).
- Key result
- Run / experiment:
- other: mean of three experiments (GARDskin)
- Parameter:
- other: Decision Value
- Value:
- 0
- 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: mean of three experiments (GARDpotency)
- Parameter:
- other: Decision Value
- Value:
- 0
- 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:
Please refer to Table 1 (Acceptance criteria for toxicity) in box "Any other information on results incl. tables".
MAIN STUDY RESULTS:
In the present study the test item was dissolved in DMSO at a concentration of 500 mM. For the Input finding assay, stock solutions with concentrations ranging from 50 mM to 1 mM were prepared by serial dilution (finally 50 µM to 1 µM). 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 RV90 ± 5% value could be determined at a test item concentration of 10 µM (10 mM stock concentration). The first main stimulation was done with a test item concentration of 10 µM (10 mM stock concentration). Cells were incubated with the test item for 24 h at 37 °C. After exposure one part of the cells were stained with propidium iodide and cell viability was measured by FACS analysis and the second part of the cells were transferred into TRIzol and stored at -80 °C.
The first main stimulation did not fulfil the cytotoxicity criteria for the test item, but for the control samples. The stimulation was repeated (first main stimulation repetition) with a separate prepared test item sample and with a small dilution range with three different concentrations (20 mM, 15 mM, 10 mM stock concentrations) because of the strength of the toxicity curve. The second and third main stimulation was done with the same three concentrations as in the first main stimulation repetition. Based on the results the stock concentration of 15 mM (finally 15 µM) of the first main stimulation repetition, second and third main stimulation was chosen for RNA isolation. No samples were used for isolation from the first main stimulation. The RNA samples were shipped temperature monitored by World Courier to Test Site 1. All samples arrived without any issues.
The RNA quality was measured first for all single tube TRIzol isolations. The positive control sample of the second experiment did not fulfil the quality criteria and the pooled TRIzol sample was measured. This pooled sample fulfilled the quality criteria. This sample was used for further nanostring measurement. The nanostring measurement fulfilled all the criteria and the transferred “rlf files” were used for data analysis. Two deviations to the phaseplan at SenzaGen were mentioned for the GARDskin measurement. Two RNA concentrations were calculated wrong before the nanostring measurement was performed. That is why we excluded these medium control samples from our GARDskin analysis, which do not lead to any differences in the decision values and did not influence the outcome of the test.
The decision values for the test item are > 0. Therefore the test item has to be classified as a sensitiser.The decision values of the positive control are >0 (10.17; 9.37; 10.91) and for the negative control are <0 (-1.57; -1.56; -1.43). The controls confirmed the validity of the study for all experiments as shown in Table 1 in box "Any other information on results incl. tables". Due to a sensitising potential the potency of the test item was measured. Based on the results, the test item is categorized into class 1A as the decision values for the test item are > 0. The decision values of the positive control are (8.36, 8.69, 8.72), which is class 1A.
For individual decision values of the test item and the controls, see Tables 2&3 in box "Any other information on results incl. tables" for the GARDskin and GARDpotency test, respectively. - Interpretation of results:
- Category 1A (indication of significant skin sensitising potential) based on GHS criteria
- Conclusions:
- In this study under the given conditions the test item did change the genomic profile of the cells for sensitisation in at least three independent experiment runs. Therefore, the test item can be considered as sensitiser and sub-categorisation into class 1A is warranted.
- Executive summary:
In the in vitro GARD^TM assay conducted with 5’-O-(4,4’-Dimethoxytrityl)thymidine (purity 97.8%) in DMSO, the sensitisation potential of the test item is based on the analysis of the relative expression levels of a biomarker signature of 196 genes using a human myeloid leukemia cell line called SenzaCell. The GARD^TM is based on chemical stimulation of the SenzaCell line, acting as an in vitro model of human Dendritic Cells (DCs). The readout of the assay is a transcriptional quantification of the genomic predictors, collectively termed the GARD^TM Prediction Signature (GPS), using Nanostring nCounter technology.
In the current study, the cells were incubated with the test item with three different concentrations (10, 15 and 20 µM) for 24 h at 37 °C. After exposure one part of the cells were stained with propidium iodide and cell viability was measured by FACS analysis and the second part of the cells were transferred into TRIzol and stored at -80 °C. Based on the results, cells treated with 15 µM were chosen for RNA isolation and subsequent analysis.
In the GARD skin analysis the decision values for the test item were >0 (4.33, 4.40 and 6.51). Thus, the test item has be classified as a skin sensitiser.
Due to a sensitising potential the potency of the test item was measured. Based on the results, the test item can be sub-categorised into class 1A as the decision values for the test item are > 0 (2.34, 1.15 and 0.60). The positive and negative controls confirmed the validity of the study.
In summary, the test item did change the genomic profile of the cells for sensitization in three independent experiment runs. Therefore, the test item can be considered as sensitiser and sub-categorised into class 1A.
- Endpoint:
- skin sensitisation: in vivo (LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2019-10-23 to 2019-12-02
- 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), 10-11 weeks (main study)
- Weight at study initiation: 19.0-19.5 (pre-test), 17.7-24.3 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 (%): 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% (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 was conducted on the ear weights 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, the Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers. - 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. - 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.
- Endpoint:
- skin sensitisation: in chemico
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2018-07-05 to 2018-09-05
- 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 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 acetonitrile, dist. water, dist. water:acetonitrile 1:1 (v/v), isopropanol, 1,4-butanediol, ethanol and tert. Butanol. The test item was completely soluble in methanol and N,N-dimethylformamide, therefore, methanol was chosen as suitable vehicle for the main experiments.
Experimental Procedure
Incubation of the Test Item with the Cysteine and Lysine Peptide
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. After 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.
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 65.83%.
- Key result
- Parameter:
- other: Mean percent of peptide depletion
- Value:
- 0.8
- 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". - Interpretation of results:
- study cannot be used for classification
- Conclusions:
- In this study (performed in accordance with OECD guideline 442C) under the given conditions 5’-O-(4,4’-Dimethoxytrityl)thymidine showed minimal reactivity (as indicated by peptide depletion) towards both cysteine and lysine peptides. However, due to the observed precipitation 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 5’-O-(4,4’-Dimethoxytrityl)thymidine was dissolved in methanol. Based on its molecular weight of 544.59 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).
From the 100 mM solution of the test item precipitation was observed immediately after diluting the test item solution into the peptide solutions and after 24 h ± 2 h incubation. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item-incubated samples to the corresponding reference control C (RC Cmethanol).
The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was 0.80%, which is below the threshold level of 6.38% based on the prediction model using the mean cysteine (1:10) as well as lysine (1:50) percent peptide depletion values. However, since precipitations were observed, a test item concentration of 100 mM as well as the full contact of peptide and test item is not guaranteed. Therefore no prediction can be made.
Referenceopen allclose all
Table 1: Induction of Luciferase Activity – Overall Induction
Overall Induction |
Concentration [µM] |
Relative Fold Induction (Relative to solvent control) |
Significance |
|||
Experiment 1 |
Experiment 2 |
Mean |
SD |
|||
Solvent Control |
- |
1.00 |
1.00 |
1.00 |
0.00 |
|
Positive Control |
4.00 |
1.19 |
1.14 |
1.16 |
0.04 |
|
8.00 |
1.48 |
1.16 |
1.32 |
0.23 |
|
|
16.00 |
1.53 |
1.32 |
1.42 |
0.15 |
|
|
32.00 |
2.14 |
1.68 |
1.91 |
0.33 |
|
|
64.00 |
3.29 |
2.72 |
3.01 |
0.40 |
* |
|
Test Item |
0.98 |
1.06 |
1.10 |
1.08 |
0.03 |
|
1.95 |
1.26 |
1.06 |
1.16 |
0.14 |
|
|
3.91 |
1.48 |
1.29 |
1.38 |
0.13 |
|
|
7.81 |
1.22 |
0.83 |
1.03 |
0.28 |
|
|
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 compared to solvent control according to Student’s t-test, p<0.05
In the present study 5’-O-(4,4’-Dimethoxytrityl)thymidine was dissolved in DMSO. For the dose finding assay concentrations ranging from 7.81 µg/mL to 1000 µg/mL (experiment 1) and from 1.95 µg/mL to 250 µg/mL (experiment 2) 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 13.60 ± 2.98 µg/mL was derived in the dose finding assay. Based on the CV75, the main experiment was performed covering the following concentration steps: 16.32, 13.60, 11.33, 9.44, 7.87, 6.56, 5.46, 4.55 µg/mL
In the dose finding experiment 1 precipitation was observed for the highest three concentrations and turbidity was observed for the highest four concentrations of the test item when mixing the test item stock solutions with cell culture medium. In the dose finding experiment 2 precipitation was observed for the highest concentration and turbidity was observed for the highest four concentrations of the test item when mixing the test item stock solution with cell culture medium. Sonification was used to aid solubilisation.
Cells were incubated with the test item for 24 h at 37 °C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.
Cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at thehighest test item concentration was reduced to 61.9% (CD86), 61.5% (CD54) and 60.6% (isotype IgG1 control) in the first experiment and to 58.4% (CD86), 58.0% (CD54) and 57.8% (isotype IgG1 control) in the second experiment.
The expression of the cell surface marker CD86 was upregulated above the threshold of 150% to 214%, 247%, 245%, 235%, 204%, 199% and 164% in the first experiment and to 314%, 328%, 336%, 358%, 289%, 320%, 292% and 267% in the second experiment. The upregulation above the threshold of 150% was observed starting from a concentration of 5.47 µg/mL to 16.32 µg/mL in the first experiment and starting from a concentration of 4.55 µg/mL to 16.32 µg/mL in the second experiment.
The expression of the cell surface marker CD54 was upregulated to 991%, 641%, 410%, 300% and 217% in the first experiment and to 977%, 615%, 417%, 285% and 206% in the second experiment. The upregulation above the threshold of 200% was observed starting from a concentration of 7.87 µg/mL to 16.32 µg/mL in both experiments.
The EC150 value was calculated with 4.88 µg/mL and the EC200 value was calculated with 7.63 µg/mL. Since only two independent runs are performed, the higher EC150 or EC200 of the two calculated values was adopted. The EC values could potentially contribute to the assessment of sensitising potency when used in integrated approaches such as IATA.
Since the expression of both cell surface markers clearly exceeded the threshold in two independent experiments the test item is 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. |
Cell Viability [%] |
Mean Fluorescence Intensity |
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 |
- |
96.2 |
95.5 |
95.9 |
2299 |
1072 |
536 |
1763 |
536 |
82 |
87 |
429 |
200 |
Solvent Control |
0.20% |
95.9 |
95.7 |
95.8 |
2678 |
1144 |
531 |
2147 |
613 |
100 |
100 |
504 |
215 |
DNCB |
4.00 |
83.6 |
82.4 |
82.6 |
7768 |
2753 |
587 |
7181 |
2166 |
334 |
353 |
1323 |
469 |
5’-O-(4,4’-Dimethoxytrityl)thymidine |
16.32 |
61.9 |
61.5 |
60.6 |
5259 |
6744 |
671 |
4588 |
6073 |
214 |
991 |
784 |
1005 |
Table 2: CD54 and CD86 Expression Experiment 2
Sample |
Conc. |
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 |
- |
97.2 |
96.6 |
96.9 |
2418 |
1156 |
624 |
1794 |
532 |
103 |
93 |
388 |
185 |
Solvent Control |
0.20% |
97.9 |
97.4 |
97.2 |
2406 |
1228 |
656 |
1750 |
572 |
100 |
100 |
367 |
187 |
DNCB |
4.0 |
82.9 |
81.3 |
79.9 |
6663 |
1998 |
593 |
6070 |
1405 |
347 |
246 |
1124 |
337 |
5’-O-(4,4’-Dimethoxytrityl)thymidine |
16.32 |
58.4 |
58.0 |
57.8 |
6229 |
6324 |
736 |
5493 |
5588 |
314 |
977 |
846 |
859 |
Table 1: Acceptance criteria for toxicity
Acceptance Criteria: |
Main Stimulation 1 repetition |
Main Stimulation 2 |
Main Stimulation 3 |
||||
Medium |
total ≥ 84.5 % |
93.6 |
pass |
95.7 |
pass |
95.3 |
pass |
DMSO |
RV ≥ 95.5% |
102.2 |
pass |
101.4 |
pass |
98.7 |
pass |
Positive Control |
RV 84.5% - 95.4% |
91.3 |
pass |
87.3 |
pass |
86.0 |
pass |
Table 2: GARDskin - Decision Values of the Test Item and the Controls
| Negative Control | Positive Control | 5´-O-(4,4´-Dimethoxytrityl)thymidine | |||
| Main Stimulation | Decision Values | Main Stimulation | Decision Values | Main Stimulation | Decision Values |
| 1 repetition | -1.57 | 1 | 10.17 | 1 | 4.33 |
| 2 | -1.56 | 2 | 9.37 | 2 | 4.40 |
| 3 | -1.43 | 3 | 10.91 | 3 | 6.51 |
| Mean | -1.52 | Mean | 10.15 | Mean | 5.08 |
| Predicition | |||||
| negative | positive | positive | |||
Table 3: GARDpotency -Decision Values of the Test Item and the Controls
| Positive Control | 5´-O-(4,4´-Dimethoxytrityl)thymidine | ||
| Main Stimulation | Decision Values | Main Stimulation | Decision Values |
| 1 repetition | 8.36 | 1 | 2.34 |
| 2 | 8.69 | 2 | 1.15 |
| 3 | 8.72 | 3 | 0.60 |
| Mean | 8.59 | Mean | 1.36 |
| Predicition | |||
| 1A | 1A | ||
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 |
Table 4: Depletion of the Cysteine Peptide
Sample |
Peak Area |
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 |
16.1210 |
0.4884 |
1.96 |
1.58 |
0.35 |
22.07 |
16.2340 |
0.4918 |
1.27 |
||||
16.1940 |
0.4906 |
1.51 |
Table 5: Depletion of the Lysine Peptide
Sample |
Peak Area |
Peptide Conc. [mM] |
Peptide Depletion [%] |
Mean Peptide Depletion [%] |
SD of Peptide Depletion [%] |
CV of Peptide Depletion [%] |
Positive Control |
5.8010 |
0.1970 |
60.84 |
60.13 |
0.81 |
1.35 |
5.8790 |
0.1996 |
60.31 |
||||
6.0370 |
0.2050 |
59.25 |
||||
Test Item |
15.3040 |
0.5192 |
0.00 |
0.01 |
0.02 |
173.21 |
15.1130 |
0.5127 |
0.00 |
||||
14.9870 |
0.5085 |
0.04 |
Table 6: Prediction of sensitising potential of the test item
Prediction Model |
|||
Test Substance |
Mean Peptide Depletion [%] |
Reactivity Category |
Prediction |
Test Item |
0.80 |
Minimal Reactivity |
negative |
Positive Control |
65.83 |
High Reactivity |
positive |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not sensitising)
- Additional information:
The skin sensitisation potential and potency of the substance 5’-O-(4,4’-dimethoxytrityl)thymidine was assessed in three skin sensitisation assays (OECD 442D, OECD 442E and GardTM assay), one in chemico assay (OECD 442C) and in one in vivo LLNA study (OECD 429).
In the first study, conducted according to OECD 442E with the substance in DMSO, 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 a 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 a third study, protein depletion was analysed in accordance with OECD guideline 442C. Due to low solubility of the test item it was not possible to conduct the OECD 442C assay. Due to the observed precipitation, the prediction model does not apply, and a prediction could not be made.
In a fourth study the test item was tested in the GARD^TM assay. Based on the results from the GARDskin biomarker set, the test item can be considered to be a sensitiser. In addition, based on the results from the GARDpotency genomic biomarker set, sub-categorisation into Cat 1A is applicable.
In addition to the contradictory in vitro/in chemico data, suitable information from an in vivo study conducted according to OECD 429 was used to assess the skin sensitising potential of the target substance. 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.
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.
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