N,N-bis(2-hydroxyethyl)undec-10-enamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 24, 2017 to January 26, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: 442E (human cell line activation test (h-CLAT)

Deviations:

yes

Remarks:

see 'Principles of method if other than guideline'

Principles of method if other than guideline:

In the preliminary experiment the cell pellets were resuspended in 400 µL washing buffer and were not blocked with 600 µL of blocking solution (washing buffer containing 0.01% (w/v) globulin (Cohn fraction II, III, Human)) and incubated at 4°C for 15 minutes as stated in the Study Plan.
This minor deviation did not affect the validity and integrity of the scientific results obtained during this study.

GLP compliance:

yes (incl. QA statement)

Type of study:

other: human cell line activation test (h-CLAT)

Test material

Test material information

Constituent 1

Reference substance name:

N,N-bis(2-hydroxyethyl)undec-10-enamide

EC Number:

262-114-9

EC Name:

N,N-bis(2-hydroxyethyl)undec-10-enamide

Cas Number:

60239-68-1

Molecular formula:

C15H29NO3

IUPAC Name:

N,N-bis(2-hydroxyethyl)undec-10-enamide

impurity 1

Reference substance name:

2,2'-iminodiethanol

EC Number:

203-868-0

EC Name:

2,2'-iminodiethanol

Cas Number:

111-42-2

Molecular formula:

C4H11NO2

IUPAC Name:

2,2’-iminodiethanol

Test material form:

liquid

In vitro test system

Details on the study design:

Preparation of cells:

The human monocytic leukaemia cell line, THP-1 (TIB-202, ATCC) was used for performing the h-CLAT assay.
For testing, THP-1 cells were seeded at a density of either 0.1+E6 cells/mL or 0.2+E6 cells/mL, and pre-cultured in culture flasks for 72 or 48 h, respectively. It is important that the cell density in the culture flask just after the pre-culture period is as consistent as possible in each experiment, because the cell density in the culture flask just after pre-culture could affect the CD86/CD54 expression induced by allergens. On the day of testing, cells harvested from the culture flask was resuspended with fresh culture medium at 2+E6 cells/mL. Then, cells were distributed into a 24-well flat-bottom plate with 500 µL per well (1+E6 cells/well).

Dose finding assay:
A dose finding assay was performed to determine the CV75, being the test substance concentration that results in 75% cell viability (CV) compared to the solvent/vehicle control. The CV75 value was used to determine the concentration of test substance for the CD86/CD54 expression measurement.

Preparation of test and control substances:
Final range of concentrations of THP-1 cell suspension in the plate was of 7.81 - 1000 µg/mL. The solvent was tested at a single final concentration in the plate of 0.2%.

Application of the test and control substances:
The culture medium or working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well flat-bottom plate. The treated plates were incubated for 24 ± 0.5 hours at 37°C under 5% CO2.

Preparation of the test and control substances:
The test substance was first diluted to the concentration corresponding to 500-fold of the 1.2 × CV75 determined in the dose finding assay. Then, 1.2-fold serial dilution was made using the corresponding solvent/vehicle to obtain the dilutions (eight concentrations ranging from 500 x 1.2 x CV75 to 500 x 0.335 x CV75 were tested in the h-CLAT assay. The dilutions were then further diluted 250-fold into the culture medium (working solutions). These working solutions were further diluted two-fold for use for exposure in the plate. Only 24-well plates were used for CD86/CD54 expression measurement. The solvent/vehicle control was prepared. DNCB (2,4-dinitrochlorobenzene) was used as the positive control for CD86/CD54 expression measurement at a final single concentration of 4.0 µg/mL in the plate.

Application of test and control substances:
Test and control substances prepared as working solutions (500 µL) were mixed with 500 µL of suspended cells (1 +E6 cells) at 1:1 ratio, and cells were incubated for 24 ± 0.5 h. In each run, a single replicate for each concentration of the test and control substances were sufficient because a prediction was obtained from at least two independent runs.

CELL STAINING ANALYSIS:
After 24 ± 0.5 h of exposure, cells were transferred from the 24-well plate into sample tubes, collected by centrifugation (at 250 g for 5 min at 4°C), and then washed with washing buffer. After washing, cells were blocked with 600 µL of blocking solution (washing buffer containing 0.01% (w/v) globulin (Cohn fraction II, III, Human) and incubated at 4°C for 15 min. After blocking, cells were divided into three aliquots of 180 µL into a 96-well round-bottom plate or microtube. After centrifugation, cells were stained with 50 µL of FITC-labelled anti-CD86, anti-CD54, or mouse IgG1 (isotype) antibodies at 4°C for 30 min. The antibodies described in the h-CLAT DB-ALM protocol no. 158 were used (3:25 (v/v, for CD86 (BD-PharMingen, #555657; Clone: Fun-1)) or 3:50 (v/v, for CD54 (DAKO, #F7143; Clone: 6.5B5) and IgG1 (DAKO, #X0927) with staining buffer).

After 24 ± 0.5 h of exposure 250 µL of each cell-preparation was transferred into a 96-well round-bottom plate and cells were collected by centrifugation (at 250 g for 5 min at 4°C). The supernatants were discarded and the remaining cells were washed twice with washing buffer (0.1% BSA in DPBS). Cell pellets were resuspended in 400 µL washing buffer. Shortly prior to analysis 20 µL propidium iodide solution (12.5 µg/mL) were added (final concentration of PI: 0.6 µg/mL).

After washing with 200 µL of washing buffer twice, cells were resuspended in 400 µL washing buffer and stored until analysis at 4°C. Shortly prior to analysis, 13 µL of PI solution (final concentration of approximately 0.6 µg/mL) were added. The expression levels of CD86 and CD54, and cell viability were analysed using flow cytometry.

The propidium iodide (PI) uptake was analysed using flow cytometry with the acquisition channel FL-3 (620 nm). A total of 10 000 living cells (PI negative) were acquired. The cell viability was calculated using by the cytometer analysis program.

Data evaluation
The expression of CD86 and CD54 was analysed with flow cytometry with the acquisition channel FL-1 (525 nm). Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 positive control cells and test substance-treated cells were calculated according to the following equation:

"RFI=" "MFI of test substance-treated cells - MFI of test substance-treated isotype control cells x100 " /"MFI of solvent/vehicle-treated cells - MFI of solvent/vehicle-treated isotype control cells"

The cell viability of the isotype control cells (which are stained with mouse IgG1 (isotype) antibodies) were also calculated

Interpretation of results and prediction model
For CD86/CD54 expression measurement, each test substance was tested in at least two independent runs to derive a single prediction (positive or negative). An h-CLAT prediction was considered positive if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs, otherwise the h-CLAT prediction was considered negative:
The RFI of CD86 is equal to or greater than 150% at any tested concentration (with cell viability ≥ 50%);
The RFI of CD54 is equal to or greater than 200% at any tested concentration (with cell viability ≥ 50%).

Based on the above conditions, if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered positive and a third run does not need to be conducted. Similarly, if the first two runs are negative for both markers, the h-CLAT prediction is considered negative (with due consideration of the provisions in section 6.3 regarding negative results) without the need for a third run. The first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction was based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 (hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third run is required. If this third run is negative for both markers (hereinafter referred to as N), the h-CLAT prediction is considered negative. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered positive.

For the test substances predicted as positive with the h-CLAT, optionally, two Effective Concentrations (EC) values, the EC150 for CD86 and EC200 for CD54, i.e. the concentration at which the test substances induced a RFI of 150 or 200, may be determined. These EC values potentially could contribute to the assessment of sensitising potency when used in integrated approaches such as IATA (Integrated Approach to Testing and Assessment). They can be calculated by the following equations:

EC150 (for CD86) = B concentration + [(150 - B_RFI / A_RFI - B_RFI) x (A_concentration - B_concentration)

EC200 (for CD54) = B concentration + [(200 - B_RFI) / (A_RFI - B_RFI) x (A_concentration - B_concentration)

where,
A concentration is the lowest concentration in µg/mL with RFI > 150 (CD86) or 200 (CD54)
B concentration is the highest concentration in µg/mL with RFI < 150 (CD86) or 200 (CD54)
ARFI is the RFI at the lowest concentration with RFI > 150 (CD86) or 200 (CD54)
BRFI is the RFI at the highest concentration with RFI < 150 (CD86) or 200 (CD54)

EC150 and EC200 can be calculated based on two independent runs (if just two runs were sufficient for the prediction of skin sensitation). For the purpose of more precisely deriving the EC150 and EC200 values, a third independent run for CD86/CD54 expression measurement can be performed if required by the Sponsor. The final EC150 and EC200 values are then determined as the median value of the ECs (in case of three runs) or the higher EC150 or EC200 of the two calculated values (in case of two runs).

Acceptance criteria
The following acceptance criteria should be met when using the h-CLAT assay.
- The cell viabilities of medium and solvent/vehicle controls should be higher than 90%.
- In the solvent/vehicle control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%). RFI values of the solvent/vehicle control are calculated by using the formula ("MFI of test substance" should be replaced with "MFI of solvent/vehicle", and "MFI of solvent/vehicle" should be replaced with "MFI of (medium) control").
- For both medium and solvent/vehicle controls, the MFI ratio of both CD86 and CD54 to isotype control should be > 105%.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥ 150 and CD54 RFI ≥ 200) and cell viability should be more than 50%.
- For the test substance, the cell viability should be more than 50% in at least four tested concentrations in each run.

Negative results are acceptable only for test substances exhibiting a cell viability of less than 90% at the highest concentration tested (i.e. 1.2 x CV75 according to the serial dilution scheme. If the cell viability at 1.2 x CV75 is equal to or above 90%, the negative result should be discarded. In such a case it is recommended to try to refine the dose selection by repeating the CV75 determination. It should be noted that when 1000 µg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test chemical, a negative result is acceptable even if the cell viability is above 90%.

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: range of: 36.1- 129.3 µg/mL

Parameter:

other: RFI CD54

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks on result:

other: MFI relative to IgG [%]: >105

Results 2

Key result

Run / experiment:

other: range of: 36.1- 129.3 µg/mL

Parameter:

other: RFI CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks on result:

other: MFI relative to IgG [%]: >105

Results 3

Key result

Run / experiment:

other: Mean of IgG, CD54 and CD86 [%]

Parameter:

other: Viability

Value:

50

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks on result:

other: >50% in at least 4 concentrations

Other effects / acceptance of results:

- A dose finding assay was performed to determine the CV75, being the test substance concentration that results in 75% cell viability (CV) compared to the solvent/vehicle control.
- The cell viability of the isotype control cells (which are stained with mouse IgG1 (isotype) antibody) was calculated and was >105%.
- The h-CLAT prediction was considered negative as:
The RFI of CD86 was below 150% at any tested concentration (with cell viability ≥50%);
The RFI of CD54 was below 200% at any tested concentration (with cell viability ≥50%).

The vehicle control and the positive control DNCB were run in both experiments. All quality criteria required were fulfilled.

Any other information on results incl. tables

For detailed results tables kindly refer to the attached background materials section of the IUCLID.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance did not reveal any sensitising properties in the h-CLAT test method.

Executive summary:

A study was conducted to evaluate the skin sensitisation potential of the test substance, C11-unsatd. DEA (90.6% active), according to OECD Guideline 422E, in compliance with GLP. The h-CLAT assay is an in vitro assay that quantifies changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukaemia cell line, THP-1 cells, following 24 h exposure to the test substance. These surface molecules are typical markers of monocytic THP-1 activation and may mimic DC activation, which plays a critical role in T-cell priming. The changes of surface marker expression were measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement was also conducted concurrently to assess whether upregulation of surface marker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to solvent/vehicle control were calculated and used in the prediction model to support the discrimination between sensitisers and non-sensitisers. The test substance was dissolved in DMSO. A correction factor of 1.104 was used was used due to the purity of the test substance (90.60% Amide). A dose finding assay was performed to determine the CV75, being the test substance concentration that results in 75% cell viability (CV) compared to the solvent/vehicle control. Eight dilutions (eight concentrations) were prepared, by two-fold serial dilution with DMSO and a final range of concentrations in the plate of 7.81 - 1000 µg/mL medium. DMSO was used as solvent control tested at a single final concentration in the plate of 0.2%. The CV75 value was used to determine the concentration of test substance for the CD86/CD54 expression measurement. In this preliminary experiment (consisting of two independent runs) an average CV75 of 107.7 µg/mL and a maximum test concentration for the main experiment of 129.3 µg/mL was calculated. Hence, PC-2017-721 was tested at 8 concentrations in the range from 36.1 to 129.3 µg/mL. DNCB (2,4-dinitrochlorobenzene) was used as the positive control for CD86/CD54 expression measurement at a final single concentration of 4.0 µg/mL in the plate. Each experiment consisted of two independent runs for CD86/CD54 expression measurement.The expression of CD86 and CD54 was analysed with flow cytometry with the acquisition channel FL-1 (525 nm). Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 of the medium, the positive control cells and the test substance-treated cells were calculatedcompared to the solventcontrol. The cell viability of the isotype control cells (which are stained with mouse IgG1 (isotype) antibody) was also calculated.The vehicle control and the positive control DNCB were run in both experiments. All quality criteria for the vehicle control and the positive control required were fulfilled. The h-CLAT prediction was considered negative as: the RFI of CD86 was below 150% at any tested concentration (with cell viability ≥50%) and the RFI of CD54 was below 200% at any tested concentration (with cell viability ≥50%). Under the study conditions, the test substance did not reveal any sensitising properties in the h-CLAT test method (Spruth, 2018).