3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile

Endpoint summary

Description of key information

The skin sensitisation potential of the target substance 2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite (99.6% purity) was assessed in two in vitro skin sensitisation studies conducted according to OECD 442D and OECD 442E. In both in vitro studies the target substance showed no skin sensitising potential. However, in a dermal sensitisation study conducted equivalent to OECD guideline 429 with the target substance dissolved in AOO (4:1 (v/v) acetone / olive oil), the test item was tested positive for skin sensitization.

By applying the weight-of-evidence approach, it can be concluded that the substance 2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite is considered to be a skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-12-11 to 2018-06-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

adopted: February 04, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The induction of the Keap1-Nrf2-ARE signalling pathway by small electrophilic substances such as skin sensitisers was reported by several studies and represents the second key event of the skin sensitisation process as described by the AOP. Therefore, the KeratinoSens™ assay is considered relevant for the assessment of the skin sensitisation potential of chemicals.

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dissolved in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity ≥99%; AppliChem; Lot No.: 0001055932, 0000978834). A stock solution of 200 mM was prepared by pre-weighing the test material into a glass vial. Vortex mixing was used to aid solubilisation.
Based on the DMSO stock solution, serial dilutions are made using the solvent (DMSO) to obtain 12 master concentrations of the test item (0.098 to 200 mM). The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the master solutions were further diluted 1:25 in cell culture medium.
These 1:25 diluted test item solutions were finally diluted 1:4 when incubated with the cells so that the final concentrations of the tested chemical range from 0.98 to 2000 µM. Based on this procedure the final concentration of the solvent (DMSO) was 1% (v/v) in all test item concentrations and controls.

Details on the study design:

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

CELL LINE:
The test was carried out using the transgenic cell line KeratinoSens™ (Givaudan, Switzerland), a cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and were used for routine testing. Only cells at a low passage number <25 (passage 3 in experiment 1; passage 5 in 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 item exposure, cells were cultured in medium in test item exposure medium.

LUCIFERASE ASSAY SYSTEM:
The luciferase activity was determined using the following products purchased from Promega. All components were used according to the instructions of the manufacture manual. The kit (Promega, Cat. No.: E1501, Lot No.: 0000268778) 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 30 mL of Luciferase Cell Culture Lysis 5x Reagent.
Prior to use lysis buffer was diluted 1:5 with dist. water (Sigma; Lot No.: RNBF7110).

DOSE GROUPS:
Negative Control: 1% (v/v) DMSO in test item exposure medium
Positive Control: CA: 4 µM, 8 µM, 16 µM, 32 µM, 64 µM
Test Item: 12 concentrations of the test item: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 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 96-well plate in every independent run.

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

LUCIFERASE ACTIVITY:
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 µL of 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 1000 ms before assessing the luciferase activity for 2000 ms. This procedure was repeated for each individual well of the 96 well-plate.

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

DATA ANALYSIS:
For each test item two independent repetitions using separately prepared test item solutions and independently harvested cells are necessary to derive a prediction. Each independent run consisted of three replicates for every concentration step of the test item and the positive control. In case of discordant results, a third independent run is performed
PREDICTION MODEL:
The test item is considered positive in accordance with UN GHS “Category 1” for skin sensitisation if the following conditions were met in at least two independently prepared test runs:

- Imax is >1.5-fold increased and statistically significant (p <0.05) compared to the negative control;
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5;
- EC1.5 value is <1000 µM;
- an apparent overall dose-response relationship for luciferase induction.

If in a given run, all of the three first conditions are met but a clear dose-response relationship for the luciferase induction cannot be observed, the result of that run 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 (2.46 in experiment 1; 5.40 in experiment 2).

Run / experiment:

other: Mean of experiment 1 and 2

Parameter:

other: max luciferase activity (Imax) induction

Value:

1.12

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: max luciferase activity (Imax) induction

Value:

1.16

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: max luciferase activity (Imax) induction

Value:

1.09

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
- For each test item two independent repetitions using separately prepared test item solutions and independently harvested cells are necessary to derive a prediction. Each independent run consisted of three replicates for every concentration step of the test item and the positive control. In case of discordant results, a third independent run is performed.
- For every concentration showing >1.5-fold luciferase activity induction, statistical significance (p< 0.05) was calculated using a two-tailed Student’s t-test comparing the luminescence values for the three replicated samples with the luminescence values in the solvent (negative) control wells.
- The lowest concentration with >1.5-fold luciferase activity induction was the value determining the EC1.5 value. It was checked in each case whether this value was below the IC30 value, indicating that there was less than 30% reduction on cellular viability at the EC1.5 determining concentration.

ACCEPTANCE OF RESULTS:
The acceptance criteria proposed by the OECD test guideline 442D were met in this test.

- In the first experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.
- In the second experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5value could be calculated. Within the second experiment the two lowest concentrations showed cytotoxic effects (viability <70%). Since the higher concentrations showed no cytotoxic effects and no dose effect relationship could be observed, this observation did no influence the evaluation. This effect may be caused by a plate effect or by a technical error during the seeding of cells.
- No dose-response relationship for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.


For individual results see Table 1 in box 'Any other information on results incl. tables'.

Table 1: Induction of Luciferase Activity - Overall induction

Overall Induction	Concentration [µM]	Relative Fold Induction^1)
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	1.00	1.00	1.00	0.00
Positive Control	4.00	0.97	1.09	1.03	0.08
	8.00	1.07	1.23	1.15	0.11
	16.00	1.41	1.47	1.44	0.04
	32.00	1.63	2.20	1.92	0.41
	64.00	2.46	5.40	3.93	2.08
Test Item	0.98	1.11	0.95	1.03	0.11
	1.95	1.16	0.90	1.03	0.18
	3.91	1.14	0.87	1.00	0.19
	7.81	1.09	0.87	0.98	0.15
	15.63	1.06	0.87	0.97	0.13
	31.25	1.08	0.91	1.00	0.11
	62.50	1.00	0.95	0.98	0.04
	125.00	1.14	0.94	1.04	0.14
	250.00	1.06	1.08	1.07	0.02
	500.00	1.06	1.01	1.03	0.04
	1000.00	0.99	1.09	1.04	0.07
	2000.00	0.96	0.97	0.97	0.01

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

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

Interpretation of results:

GHS criteria not met

Conclusions:

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

Executive summary:

In a dermal sensitisation study conducted according to OECD 442D with 2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite (99.6% purity) in DMSO, the sensitisation potential of the test item was assessed on the basis of the activation of keratinocytes using the in vitro KeratinoSens™ cell line. Cells were incubated with the test item for 48 h at 37 °C and later checked for luciferase activity. Sensitisation was scored by measuring maximum luciferase activity induction (Imax), cell viability and EC1.5. For both experiments 1 and 2 no significant luciferase induction was observed within the tested concentration range. Therefore, no EC1.5 value could be calculated. Based on the results, the test item is considered to be a non-sensitiser.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-12-11 to 2018-06-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 442E

Version / remarks:

adopted: 09 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

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

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was freshly prepared immediately prior to use. The test item was soluble in DMSO at a concentration of 500 mg/mL.
Stock solutions were prepared by diluting the highest soluble concentration seven times with a constant dilution factor of 1:2.
The working stock solutions were prepared by diluting each stock solution 250 times with cell culture medium.
Phase separation was observed when diluted 1:250 in cell culture medium for test item solutions starting from a final assay concentration of 250 µg/mL onwards. Sonication was used to aid solubilisation.
The working stock solutions were applied to the cells by adding equal volumes of each solution to prepared cells, resulting in a further 1:2 dilution of the working solutions. The solvent (DMSO) was present at a constant volume ratio of 0.2% (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.

Details on the study design:

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

CELL LINE:
The test was carried out using THP-1 cells (ATCC® TIB-202TM), an acute human monocytic leukemic cell line used as a surrogate for DC. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and subcultured at least 2 weeks before they were used in the in vitro h-CLAT test. Cells at passage number (<30) were used. Cells are routinely passaged every 2-3 days at a density of 0.1 – 0.2 x 106 cells/mL. Cells were cultured in 75 cm2 culture flasks (Greiner) in cell culture medium consisting of Roswell Park Memorial Institute medium (RPMI-1640, Gibco Life Science; Cat. No.: 31870-025) supplemented with 10% fetal bovine serum, 25 mM HEPES, L-glutamine, 0.05 mM 2-mercaptoethanol and 100 U/mL penicillin/ 100 µg/mL streptomycin at 37 +/- 1 °C and 5% CO2.


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

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

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

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

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

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

Positive control results:

The positive control led to an upregulation of CD54 and CD86 in all experiments. The threshold of 150% for CD86 (413% experiment 1; 353% experiment 2; 293% experiment 3 ) and 200% for CD54 (608% experiment 1; 325% experiment 2; 302% experiment 3) were clearly exceeded.

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: CD86 upregulation (%)

Remarks:

at highest tested concentration of 1000 µg/mL

Value:

167

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: Experiment 1

Parameter:

other: CD54 upregulation (%)

Remarks:

at highest tested concentration of 1000 µg/mL

Value:

91

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: CD86 upregulation (%)

Remarks:

at highest tested concentration of 1000 µg/mL

Value:

93

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: CD54 upregulation(%)

Remarks:

at highest tested concentration of 1000 µg/mL

Value:

61

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Experiment 3

Parameter:

other: CD86 upregulation (%)

Remarks:

at highest tested concentration of 1000 µg/mL

Value:

118

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Experiment 3

Parameter:

other: CD54 upregulation (%)

Remarks:

at highest tested concentration of 1000 µg/mL

Value:

56

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

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

For individual results see Table 1 in "Any other information on results incl. tables".

Summary of Results:

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 96.3% (from the CD86 expression experiment), 95.7% (from the CD54 expression experiment) and 95.6% (when tested withisotype IgG1 control)compared to total number of acquired cellsin the first experiment, to 95.8% (CD86), 95.8% (CD54) and 95.9% (isotype IgG1 control) in the second experiment and to 91.7% (CD86), 91.8% (CD54) and 91.7% (isotype IgG1 control)compared to total number of acquired cellsin the third experiment.In the first experiment the expression of the cell surface marker CD86 was upregulated above the threshold of 150% to a maximum of 167% at the highest test item concentration. The expression of the cell surface marker CD54 was not upregulated above the threshold of 200%. In the second and third experiment the expression of the cell surface marker CD86 was not upregulated above the threshold of 150% and the expression of the cell surface marker CD54 was not upregulated above the threshold of 200%. Since the expression of both cell surface markers did not exceed the threshold in two independent experiments the test item is considered to be no skin sensitiser.

Table 1: CD54 and CD86 Expression Experiment 1

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			Isotype IgG1 Corrected Mean Fluorescence Intensity		Relative Fluorescence Intensity (RFI) [%]		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	96.4	96.6	96.6	1449	883	592	857	291	79	92	245	149
Solvent Control (0.2% DMSO)	-	96.8	96.7	96.6	1669	903	585	1084	318	100	100	285	154
DNCB	4.00	82.6	82.1	82.8	5066	2525	590	4476	1935	413	608	859	428
2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite	1000	96.3	95.7	95.6	2385	861	573	1812	288	167	91	416	150

 

Table 2: CD54 and CD86 Expression Experiment 2

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			Isotype IgG1 Corrected Mean Fluorescence Intensity		Relative Fluorescence Intensity (RFI) [%]		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	96.8	96.8	96.1	2399	1162	537	1862	625	85	95	447	216
Solvent Control (0.2% DMSO)	-	97.1	96.8	96.7	2710	1189	530	2180	659	100	100	511	224
DNCB	4.0	86.1	85.9	84.8	8313	2760	620	7693	2140	353	325	1341	445
2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite	1000.00	95.8	95.8	95.9	2597	978	575	2022	403	93	61	452	170

 

Table 3: CD54 and CD86 Expression Experiment 3

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			Isotype IgG1 Corrected Mean Fluorescence Intensity		Relative Fluorescence Intensity (RFI) [%]		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	C86	CD54
Medium Control	-	94.3	94.2	94.3	2933	1332	740	2193	592	78	94	396	180
Solvent Control (0.2% DMSO)	-	93.6	94.0	93.5	3512	1346	713	2799	633	100	100	493	189
DNCB	4.0	76.4	75.6	76.8	8970	2681	770	8200	1911	293	302	1165	348
2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite	1000.0	91.7	91.8	91.7	4032	1090	735	3297	355	118	56	549	148

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item 2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite did not upregulate the cell surface markers in two of three independent experiment runs. Based on these results, the test item is not considered to be a skin sensitiser.

Executive summary:

In a skin sensitisation study conducted according to OECD 442E with 2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite (99.6 % purity), the sensitisation potential of the test item was assessed on the basis of the activation of dendritic cells using the in vitro human cell line activation test (h-CLAT). Cells were incubated with the test item for 24 h at 37 °C and later checked for cell viability and expression of CD86 and CD54 cell surface markers. Sensitisation was scored by measuring cell viability and checking the expression of both cell surface markers. CD54 and CD86 were not upregulated above the threshold of 200% (CD54) and 150% (CD86) in two of three experiments. Based on these results, the test item is not considered to be a skin sensitiser under the UN GHS criteria.

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2002-09-24 to 2003-02-19

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

- Name used in the report: CE PHOSPHITE

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Supplier: Applied Biosystems, LOT: 0204061

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was prepared at concentrations of 10, 25 and 50% in 4:1 acetone/olive oil. All solutions were prepared freshly each day of dosing.

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Jackson Laboratories, Bar Harbor, ME 04609, USA
- Females (if applicable) nulliparous and non-pregnant: not specified
- Microbiological status of animals, when known:
- Age at study initiation: 7 - 8 weeks
- Weight at study initiation:18 - 22 g
- Housing: grouped 5 per cage, in compliance with the National Research Council “Guide for the Care and Use of Laboratory Animals”.
- Diet (e.g. ad libitum): Certified Rodent diet #7012C (Harlan Teklad) or equivalent, ad libitum
- Water (e.g. ad libitum): water, ad libitum
- Acclimatation period: 11 days
- Indication of any skin lesions: not specified, animals were assessed to their general health by veterinary staff or other authorized personnel.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 26
- Humidity (%): 30 to 70
- Photoperiod (hrs dark / hrs light): 12 /12
- IN-LIFE DATES: From: To: from 2002-10-20 to 2002-06-26

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Substance: 10%, 25% and 50%
Positive control: 0.1%

No. of animals per dose:

5

Details on study design:

EXPERIMENTAL DESIGN:
The test article, positive or vehicle control were applied daily (25 µL/ear) on the dorsal surface of both ears, once per day on Days 1, 2, and 3. Any irritation observed after test article application was recorded. On day 6 the mice were injected i.v. with 20 µCi of 3^H-Thymidine in 250 µL of saline. Five hours later the mice were euthanized with CO2 and the draining auricular lymph nodes removed. A single cell suspension was prepared from the lymph nodes of each mouse. Cells were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloroacetic acid (TCA) overnight at 2 -8 °C. The pellets were recovered by centrifugation and resuspended in 1mL of TCA and transferred to 10 mL of scintillation fluid. Incorporation of 3^H-thymidinewas measured in a ß-scintillation.

OBSERVATIONS:
- Morbidity/Mortality: Animals were observed daily.
- Clinical Observation: Cage side observations were made daily

METHOD OF ANALYSIS:
The mean DPM for each group was determined. Increases in 3^H-thymidine incorporation relative to vehicle-treated control were derived for each group and recorded as stimulation indices (SI). The criterion for a positive response is that one or more concentrations of a test article elicits a 3-fold or greater increase in isotope incorporation relative to the vehicle control. Mean body weights and mean differences in body weights between Days 1 and 6 were also determined.

Positive control substance(s):

other: Dinitrochlorobenzene (DNCB)

Statistics:

Evaluation of equality of means was made by a one-way analysis of variance using the F distribution to assess statistical significance using Systat (version 9.01 SYSTAT, Inc.). If statistically significant differences between the means were found, a Dunett’s test was used to determine the degree of significance from control means.

Positive control results:

The positive control, 0.1% Dinitrochlorobenzene, resulted in a stimulation index greater than 3 (5.73) indicating a positive response. This response compared to the vehicle control was also statistically significant (p ≤ 0.05).

Key result

Parameter:

SI

Remarks:

mean of five animals

Value:

1.49

Test group / Remarks:

10% test substance

Key result

Parameter:

SI

Remarks:

mean of five animals

Value:

3.3

Test group / Remarks:

25% test substance

Key result

Parameter:

SI

Remarks:

mean of five animals

Value:

9.08

Test group / Remarks:

50% test substance

Cellular proliferation data / Observations:

- CELLULAR PROLIFERATION DATA: See table 1 in box “Any other information on results incl. tables”.
- DETAILS ON STIMULATION INDEX CALCULATION: The mean DPM for each group was determined. Increases in 3^H-thymidine incorporation relative to vehicle-treated control were derived for each group and recorded as stimulation indices (SI).
- BODY WEIGHTS: Mean body weights at Days 1 and 6 and mean changes in body weights for each treatment group were compared to the vehicle control group (see table 2 in box "Any other information on results incl. tables").
- CLINICAL OBSERVATIONS: The test item did not cause any overt toxicity.

Table 1: LLNA Assay

Group	Treatment	Dose	DPM (Mean ± SEM)	SI (Test / control ratio)	Results^1
1	Acetone/Olive oil	-	715 ± 223	-	-
2	Substance	10 %	1063 ± 247	1.49	-
3		25 %	2363 ± 591	3.30	+
4		50 %	6492 ± 1203^**	9.08	+
5	DCNB (Positive control)	0.1 %	4099 ± 1497^*	5.73	+

1 test/control ratio of 3.0 or greater reprensents a positive result

* Statistically significant difference compared to the vehicle control group (P ≤ 0.05)

** Statistically significant difference compared to the vehicle control group (P ≤ 0.01)

Table 2: Body weight

Group	Treatment	Dose	Body weights (g) (Mean ± SEM)		Change in Body Weight (g) (Mean ± SEM)
			Day 1	Day 6
1	Acetone/Olive oil	-	20.0 ± 0.8	21.2 ± 1.0	1.2 ± 0.4
2	Substance	10 %	20.0 ± 0.6	21.4 ± 0.9	1.4 ± 0.2
3		25 %	20.2 ± 0.4	21.0 ± 0.4	0.8 ± 0.2
4		50 %	20.0 ± 0.7	21.0 ± 0.7	1.0 ± 0.3
5	DCNB (Positive Control)	0.1 %	19.6 ± 0.7	20.8 ± 0.5	1.2 ± 0.4

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

In conclusion, in a mouse local lymph node assay, the test item is considered to be a skin sensitizer.

Executive summary:

In a dermal sensitisation study conducted equivalent to OECD guideline 429 with CE PHOSPHITE dissolved in AOO (4:1 (v/v) acetone / olive oil), young adult female CBA/J mice (5 per dose group) were tested at concentrations of 10%, 25% and 50% in a local lymph node assay (LLNA). Dinitrochlorobenzene (0.1%) was used as a positive control. Mean body weights at Days 1 and 6 and mean changes in body weights for each treatment group were compared to the vehicle control group. No statistically significant differences in body weight were observed for any of the test article treated groups when compared to the vehicle control group. In addition, the test item did not cause any signs of toxicity during the daily clinical oberservation. The positive control did induce an appropriate response (SI= 5.73). The test item was positive at concentrations of 25 and 50% with the response at 50% being statistically significant (p< 0.01). The stimulation indices were 3.30 and 9.08, respectively. The test item was negative at a concentration of 10% (SI = 1.49).

In this study, the test item is considered to be a skin sensitiser and has to be classified as Skin Sens 1B based on CLP criteria.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitisation potential of the target substance 2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite (99.6% purity) was assessed in two in vitro skin sensitisation studies conducted according to OECD 442D and OECD 442E. Furthermore, data of an in vivo study (Local Lymph Node Assay) is available. In the first study conducted according to OECD 442D, the sensitisation potential of the test item was assessed on the basis of the activation of keratinocytes using the in vitro KeratinoSens™ cell line. Cells were incubated with the test item for 48 h at 37 °C and later checked for luciferase activity. Sensitization was scored by measuring maximum luciferase activity induction (Imax), cell viability and EC1.5. For both experiment I and II, Imax was less than a 1.5-fold increase and cell viability was greater than 70%. No significant luciferase induction was observed within the tested concentration range.

In the second study, conducted according to OECD 442E, the sensitisation potential of the test item was assessed on the basis of the activation of dendritic cells using the in vitro human cell line activation test (h-CLAT). Cells were incubated with the test item for 24 h at 37 °C and later checked for cell viability and expression of CD86 and CD54 cell surface markers. Sensitisation was scored by measuring cell viability and checking the expression of both cell surface markers. CD54 and CD86 were not upregulated above the threshold of 200% (CD54) and 150% (CD86) in two of three experiments.

In the in vivo study, young adult female CBA/J mice (5 per dose group) were tested at concentrations of 10%, 25% and 50% in a local lymph node assay (LLNA). The test item was positive at concentrations of 25 and 50% with the response at 50% being statistically significant (p< 0.01). The stimulation indices were 3.30 and 9.08, respectively. The test item was negative at a concentration of 10% (SI = 1.49).

Based on the results of the Local Lymph Node Assay and taking the weight-of-evidence approach, the test item is considered to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on a weight-of-evidence approach, 2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite is considered to be a skin sensitiser and classification for Skin Sens 1B, H317 is warranted.