1,1'-(chlorophenylmethylene)bis[4-methoxybenzene]

Endpoint summary

Description of key information

The skin sensitisation potential of the test item 4,4’-Dimethoxytrityl chloride (purity 100%) was assessed in two in vitro skin sensitisation assays (OECD 442D and OECD 442E).

In the first study conducted according to OECD 442E with the target substance in tetrahydrofuran (THF), 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). Furthermore, in a second study conducted according to OECD 442D with the target substance in DMSO, the sensitisation potential of the test item was assessed based on the activation of keratinocytes using the in vitro KeratinoSens™. Based on the results from both studies, 4,4’-Dimethoxytrityl chloride must be considered as a skin sensitzer and classification 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:

2018-01-05 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
- Treatment of test material prior to testing:
All test item solutions were freshly prepared immediately prior to use. The test item was dissolved in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity ≥99%; AppliChem; Lot No.: 0001055932). A stock solution of 200 mM was prepared by pre-weighing the test material into a glass vial. Vortex mixing was used to aid solubilisation.
Based on the DMSO stock solutions, serial dilutions were made using the solvent to obtain 12 master concentrations of the test item (0.098 to 200 mM) The stock solution of the test item were diluted eleven times using a constant dilution factor of 1:2. Then the master solutions were further diluted 1:25 in cell culture medium.
These 1:25 diluted test item solutions were diluted 1:4 in cell culture medium when incubated with the cells so that the final concentrations of the tested chemical ranged from 0.98 to 2000 µM. Based on this procedure the final concentration of the solvent (DMSO) was 1% (v/v) in all test item concentrations and controls.

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 12, experiment 1; passage 11, experiment 2) were used. Cells were cultured in 75 cm² culture flasks (Greiner) in maintenance medium at 37 +/- 1 °C and 5% CO2 in a humidified incubator. For test material exposure, cells were cultured in medium.

LUCIFERASE ASSAY SYSTEM:
The luciferase activity was determined using the following products purchased from Promega. All components were used according to the instructions of the manufacture manual. The kit (Promega, Cat. No.: E1501, Lot No.: 0000276369) consisted of the following components relevant for this study:
- 10 vials Luciferase Assay Substrate (lyophilized)
- 10 x 10 mL Luciferase Assay Buffer
If freshly prepared, Luciferase Assay Substrate was dissolved in Luciferase Assay Buffer. If thawed from -80 °C, Luciferase Assay Reagent was allowed to equilibrate to room temperature prior to use.
Luciferase Cell Culture Lysis 5x Reagent
The kit (Promega, Cat. No.: E1531, Lot No.: 0000246522) consisted of the following components relevant for this study:
- 30 mL Luciferase Cell Culture Lysis 5x Reagent
Prior to use lysis buffer was diluted 1:5 with dist. water (Sigma; Lot No.: RNBG3519)

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

EXPERIMENTAL PROCEDURE:
The incubation was performed in 96-well plates.A cell suspension of 8 × 10^4 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1× 10^4 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel, cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability.
After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 µL test item exposure medium. 50 µL of the shortly before prepared 25 times diluted master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item. All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated 96-well plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.

LUCIFERASE ACTIVITY:
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS (Gibco Life Science; Lot No.: 1877596). Subsequently 20 µL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light.
Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1.000 ms before assessing the luciferase activity for 2.000 ms. This procedure was repeated for each individual well.

CELL VIABILITY:
For the cell viability plate the medium was replaced with 200 µL test item exposure medium. 27 µL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 200 µL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5% CO2 overnight (experiment 1 and 2). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.

DATA ANALYSIS:
For every concentration showing >1.5 fold luciferase activity induction, statistical significance (p< 0.05) was calculated using a two-tailed Student’s t-test comparing the luminescence values for the three replicated samples with the luminescence values in the solvent (negative) control wells. The lowest concentration with >1.5 fold luciferase activity induction was the value determining the EC1.5 value. It was checked in each case whether this value was below the IC30 value, indicating that there was less than 30% reduction on cellular viability at the EC1.5 determining concentration.

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

If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <1000 µM is considered as inconclusive.

Positive control results:

- The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (3.36 in experiment 1; 3.17 in experiment 2).
- The calculated EC1.5 was between 7 and 34 µM (20.65 µM in experiment 1; 14.27 µM in experiment 2).

Run / experiment:

other: Mean of Experiment 1 and 2

Parameter:

other: max luciferase activity (lmax) induction

Value:

2.7

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 Experiment 1 and 2

Parameter:

other: calculated EC1.5 [µM]

Value:

7.58

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 1, at 15.62 uM

Parameter:

other: max luciferase activity (lmax) induction

Value:

1.86

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 31.25 uM

Parameter:

other: max luciferase activity (lmax) induction

Value:

3.53

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 1, at 15.62 uM

Parameter:

other: calculated EC1.5 [uM]

Value:

6.79

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 31.25 uM

Parameter:

other: calculated EC1.5 [uM]

Value:

8.36

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:

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'.

Table 1: Induction of Luciferase Activity – Overall Induction

	Concentration [µM]	Fold Induction				Significance
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	1.00	1.00	1.00	0.00
Positive Control	4.00	1.03	1.24	1.13	0.15
	8.00	1.25	1.17	1.21	0.06
	16.00	1.44	1.59	1.52	0.10	*
	32.00	1.63	2.06	1.85	0.30
	64.00	3.36	3.17	3.27	0.14	*
Test Item	0.98	1.13	1.23	1.18	0.07
	1.95	1.17	1.18	1.17	0.01
	3.91	1.11	1.21	1.16	0.07
	7.81	1.64	1.40	1.52	0.17	*
	15.63	1.86	2.84	2.35	0.70
	31.25	1.03	3.53	2.28	1.77
	62.50	0.00	0.00	0.00	0.00
	125.00	0.00	0.00	0.00	0.00
	250.00	0.00	0.00	0.00	0.00
	500.00	0.00	0.00	0.00	0.00
	1000.00	0.00	0.01	0.00	0.01
	2000.00	0.00	0.02	0.01	0.02

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

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered to be a sensitiser in accordance with UN GHS category 1.

Executive summary:

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

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

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

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-01-05 to 2018-06-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 442E

Version / remarks:

Adpoted on 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
- Treatment of test material prior to testing:
The test item was soluble in tetrahydrofuran (THF) at a concentration of 350 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.
Turbidity was observed at the four highest test item concentrations (stock solutions ranging from 43.8 – 350 mg/mL) when diluted 1:250 in cell culture medium. Sonication and warming to 37 °C were 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 (THF) 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 cm2 culture flasks (Greiner) in Roswell Park Memorial Institute medium (RPMI-1640, Gibco Life Science; Cat. No.: 31870-025) supplemented with 10% fetal bovine serum, 25 mM HEPES, L-glutamine, 0.05 mM 2-mercaptoethanol and 100 U/mL penicillin/ 100 µg/mL streptomycin at 37 +/- 1 °C and 5% CO2.

PRE-EXPERIMENTS:
Prior to testing, the quality of freshly thawed cell batch was checked by monitoring the doubling time and checking the reactivity towards 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 350 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 into a 24 well flat-bottom plate (1 x 10^6 cells/well). The solvent controls and the working solutions of the test item 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. 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 THF 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; CV75/1.2^4; 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 100-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 50-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. Moreover, the cell viability was calculated.

Positive control results:

The positive control (DNCB) led to an upregulation of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (283% experiment 1; 395% experiment 2) and 200% for CD54 (383% experiment 1; 421% experiment 2) were clearly exceeded.

Run / experiment:

other: 1

Parameter:

other: CD86 upregulation

Remarks:

at highest tested concentration of 22.74 µg/mL

Value:

204

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 22.74 µg/ml

Value:

166

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

Remarks:

at highest tested concentration 22.74 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: 2

Parameter:

other: CD54 upregulation

Remarks:

at highest tested concentration 22.74 µg/ml

Value:

248

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

In the present study, 4,4’-Dimethoxytrityl chloride was dissolved in tetrahydrofuran (THF). For the dose finding assay, concentrations ranging from 5.47 to 700 µg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37 °C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.

A CV75 of 18.95 ± 5.91 µg/mL was derived in the dose finding assay. Based on the CV75, the main experiment was performed covering the following concentration steps: 6.35, 7.62, 9.14, 10.97, 13.16, 15.79, 18.95, 22.74 µg/mL

In main experiment 1 and 2, no precipitation or turbidity of the test item was observed for all concentration steps when mixing the test item stock solutions with cell culture medium. 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 the highest test item concentration was reduced to 74.9% (CD86), 75.5% (CD54) and 73.9% (isotype IgG1 control) compared to control in the first experiment and to 71.1% (CD86), 69.9% (CD54) and 69.0% (isotype IgG1 control) compared to control in the second experiment.

- The expression of the cell surface marker CD86 was upregulated above the threshold of 150% to 204% in the first experiment and to 166% in the second experiment at the highest tested concentration at 22.74 µg/mL. The upregulation above the threshold of 150% was observed starting from a concentration of 6.35 µg/mL to 22.74 µg/mL in the first experiment and at the following concentrations of 10.97, 13.16 and 22.74 µg/mL in the second experiment.

- The expression of the cell surface marker CD54 was upregulated above the threshold of 200% to 214% in the first experiment and to 248% in the second experiment at the highest tested concentration at 22.74 µg/mL.

- The EC150 value was calculated with 10.14 µg/mL and the EC200 value was calculated with 21.68 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. [μg/mL]	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	-	95.5	95.2	95.0	2698	1071	651	2047	420	91	89	414	165
Solvent Control 2 (THF)	0.20%	95.3	95.3	95.2	2622	1235	843	1779	392	100	100	311	147
Solvent Control 1 (DMSO)	0.20%	95.4	95.8	95.2	2879	1089	619	2260	470	100	100	465	176
DNCB	4.00	83.4	83.2	82.5	7114	2508	709	6405	1799	283	383	1003	354
4,4’-Dimethoxytrityl chloride	22.74	74.9	75.5	73.9	4433	1639	800	3633	839	204	214	554	205

Table 2: CD54 and CD86 Expression Experiment 2

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI) [%]		Ratio Isotype IgG1 to [%]
		CD86	CD54	IgG Isotype	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	96.7	96.5	96.8	2270	1140	625	1645	515	95	92	363	182
Solvent Control 2 (THF)	0.20%	95.8	96.2	95.8	2340	1095	659	1681	436	100	100	355	166
Solvent Control 1 (DMSO)	0.20%	96.5	96.7	96.5	2332	1164	604	1728	560	100	100	386	193
DNCB	4.0	83.2	82.7	82.5	7543	3077	718	6825	2359	395	421	1051	429
4,4’-Dimethoxytrityl chloride	22.74	71.10	69.90	69.00	3535	1828	745	2790	1083	166	248	474	245

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In this study under the given conditions the test item 4,4’-Dimethoxytrityl chloride did upregulate the cell surface markers in two independent experiment runs. Therefore, the test item can be considered to be a skin sensitiser in accordance with UN GHS category 1.

Executive summary:

In a skin sensitisation study conducted according to OECD 442E with 4,4’-Dimethoxytrityl chloride in tetrahydrofuran (THF), 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 was upregulated above the threshold of 200% in both experiments, and CD86 was upregulated above the threshold of 200% in Experiment 1, and upregulated 166% in Experiment 2. Since one of the cell surface markers clearly exceeded the threshold in two independent experiments the test item can be considered to be a skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitisation potential of the test item 4,4’-Dimethoxytrityl chloride (purity 100%) was assessed in two in vitro skin sensitisation assays (OECD 442D and OECD 442E).

In the first study conducted according to OECD 442E with the target substance in tetrahydrofuran (THF), 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). CD54 was upregulated above the threshold of 200% in both experiments, and CD86 was upregulated above the threshold of 200% in Experiment 1, and upregulated 166% in Experiment 2. Since one of the cell surface markers clearly exceeded the threshold in two independent experiments the test item can be considered as a skin sensitizer.

Furthermore, in a second study conducted according to OECD 442D with the target substance in DMSO, the sensitisation potential of the test item was assessed based on the activation of keratinocytes using the in vitro KeratinoSens™. Sensitization was scored by measuring maximum luciferase activity induction (Imax), cell viability and EC1.5. For both experiment I and II, Imax was greater than a 1.5-fold increase, cell viability was greater than 70% and the value for EC1.5 was less than 1000 µM.

Based on the results from this study, the target substance 4,4’-Dimethoxytrityl chloride is a skin sensitizer under UN GHS “Category 1”.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In two in vitro skin sensitisation studies conducted according to OECD 442D and OECD 442E, the test item 4,4' Dimethoxytrityl chloride was tested positive for skin sensitisation. Based on the results, a classification of 4,4' Dimethoxytrityl chloride as Skin sensitiser Category 1 (H317) is warranted.