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

Endpoint summary

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:

key study

Study period:

From October 24, 2017 to January 26, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

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%.

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

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

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.

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

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).

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 18, 2017 to August 18, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

The DPRA is an in chemico method which address the molecular initiating event of the skin sensitisation AOP (Adverse Outcome Pathway), namely protein reactivity, by quantifying the reactivity of test chemicals towards model synthetic peptides containing either lysine or cysteine. Cysteine and lysine percent peptide depletion values are then used to categorise a substance in one of four classes of reactivity for supporting the discrimination between skin sensitisers and non-sensitisers.

Preparation of the cysteine or lysine-containing peptides
Stock solutions of cysteine (Ac-RFAACAA-COOH) and lysine (Ac-RFAAKAA-COOH) containing synthetic peptides of purity higher than 95% were freshly prepared just before their incubation with the test substance. The final concentration of the cysteine peptide was 0.666 mM in pH 7.5 phosphate buffer, whereas the final concentration of the lysine peptide was 0.668 mM in pH 10.2 ammonium acetate buffer.

Preparation of the test substance
Solubility of the test substance in an appropriate solvent was assessed before performing the assay. 89.66 mg of the test substance were dissolved in 3 mL acetonitrile/highly purified water 1:1 (v+v) immediately before testing to prepare a 100 mM solution. A correction factor of 1.104 was used was used due to the purity of the test substance (90.60% Amide). The test substance solution was then tested as such without any further dilution by incubating at 1:10 and 1:50 ratio with the cysteine and lysine peptides, respectively.

Positive control, reference controls and coelution control
Cinnamic aldehyde was used as positive control (PC) at a concentration of 100 mM in acetonitrile. In addition reference controls (i.e. samples containing only the peptide and added acetonitrile were also included in the HPLC run sequence and these were used to verify the HPLC system suitability prior to the analysis (reference controls A), the stability of the reference controls over time (reference control B) and to verify that the solvent used to dissolve the test substance does not impact the percent peptide depletion (reference control C). The appropriate reference control for the test substance was used to calculate the percent peptide depletion for the test substance. In addition a coelution control constituted by the test substance alone was included in the run sequence to detect possible coelution of the test substance with either the lysine or the cysteine peptide.

Incubation of the test substance with the cysteine and lysine peptide solutions
Cysteine and lysine peptide solutions were incubated in glass autosampler vials with the test substance at 1:10 and 1:50 ratio, respectively. The reaction solution was left in the dark at 25 ± 2.5°C for 24 ± 2 hours before running the HPLC analysis. The test substance assay was analyzed in triplicate for both peptides. Samples were visually inspected prior to HPLC analysis. No precipitate or phase separation was observed.

Preparation of the HPLC standard calibration curve
A standard calibration curve was generated for both the cysteine and the lysine peptides. Peptide standards were prepared in a solution of 20% acetonitrile : buffer using 100 mM sodium phosphate buffer (pH 7.5) for the cysteine peptide and 100 mM ammonium acetate buffer (pH 10.2) for the lysine peptide. Using serial dilution standards of the peptide stock solution (0.666 mM of cysteine peptide in sodium phosphate or 0.666 mM lysine peptide in ammonium acetate), 6 calibration standards were prepared to cover the range from 0.534 to 0.0167 mM. A blank of the dilution buffer was also included in the standard calibration curve. Suitable calibration curves should have an r2 > 0.99.
If a test substance promotes the oxidation of the cysteine peptide, the peak of the dimerised cysteine peptide would have been visually monitored. If dimerisation appears to have occurred, this would be noted as percent peptide depletion would be over-estimated leading to false positive predictions and/or assignment to a higher reactivity class.

Positive control results:

Cinnamic aldehyde was used as positive control at a concentration of 100 mM in acetonitrile. Treatment with the positive control substance revealed a cysteine and lysine peptide depletion of 78.34% cysteine and 57.98% lysine. These values are within the required range of 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide. The maximum standard deviation (SD) for the positive control replicates were < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion. Therefore, the study can be regarded as valid.

Key result

Parameter:

other: Mean peptide depletion (%)

Value:

1.605

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: %

Other effects / acceptance of results:

The test substance treated samples revealed a cysteine peptide depletion of 2.56% and lysine peptide depletion of 0.65% (mean peptide depletion of 1.605%) and, hence, well below 6.38% test substance is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).

The linearity of standard calibration curve was r2 = 0.9999 for cysteine peptide and 1.0000 for lysine peptide. Hence the requirement of r2 > 0.99 was met.
The mean peptide concentration of reference controls A was 0.51 mM and, hence well within the accepted range of 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C in acetonitrile/highly purified water 1:1 (v+v) was <15.0%. All acceptance criteria of validity were fulfilled in this test.

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

Conclusions:

Under the study conditions, the test substance revealed a mean cysteine and lysine peptide depletion of 1.605% and hence the test substance is considered negative and predicted to be non-sensitiser (no or minimal reactivity) in the direct peptide reactivity assay (DPRA).

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 442C, in compliance with GLP. In the DPRA in chemico method the reactivity of test substance towards model synthetic peptides cysteine and lysine is quantified. Cysteine and lysine percent peptide depletion values are used to categorise a substance in one of four classes of reactivity. Relative peptide concentration was measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm. The test substance was dissolved at a concentration of 100 mM in acetonitrile/highly purified water 1:1 (v:v). Three reference controls containing only 0.5 mM cysteine or lysine peptide solution and acetonitrile and, in addition the solvent acetonitrile/highly purified water 1:1 (v:v) were also included in the HPLC run sequence. The appropriate reference control for the test substance was used to calculate the percent peptide depletion for the test substance. Each sample was tested in triplicate. Cinnamic aldehyde was used as positive control at a concentration of 100 mM in acetonitrile. Treatment with the positive control substance revealed a cysteine and lysine peptide depletion of 78.34% cysteine and 57.98% lysine. All acceptance criteria of validity were fulfilled for the test. The test substance treated samples revealed a cysteine peptide depletion of 2.56% and lysine peptide depletion of 0.65% (mean peptide depletion of 1.605%) and, hence, well below threshold of 6.38%. Under the study conditions, the test substance revealed a mean cysteine and lysine peptide depletion of 1.605% and hence the test substance is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) in the direct peptide reactivity assay (DPRA) (Rehders, 2017).

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 18, 2017 to September 18, 2017

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: ARE-Nrf2 luciferase test method

Details on the study design:

The ARE-Nrf2 luciferase test method makes use of an immortalised adherent cell line derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The cell line contains the luciferase gene under the transcriptional control of a constitutive promoter fused with an ARE element from a gene that is known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances.

Preparation of the keratinocyte cultures:
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element was used (KeratinoSens™ cell line1). Cells were propagated and stored frozen as a homogeneous stock. Cells from this original stock were propagated up to a maximum passage number of 25 and were employed for routine testing using the appropriate maintenance medium. For testing, cells were 80-90 % confluent, and care was taken to ensure that cells were never grown to full confluence. One day prior to testing cells were harvested and distributed into 96-well plates (10 000 cells/well). Attention was paid to avoid sedimentation of the cells during seeding to ensure homogeneous cell number distribution across wells. For each repetition, three technical replicates were used for the luciferase activity measurements, and three parallel technical replicates used for the cell viability assay.

Preparation of the test and control substances:
59.8 mg of the test substance were dissolved in 1 mL dimethyl sulfoxide (DMSO) to a concentration of 200 mM. A correction factor of 1.10 was used was used due to the purity of the test item (90.60% Amide). Fresh preparations of the test and control substances were used for the treatment. The final concentration of the vehicle in the culture system did not affect cell viability or growth rate. Based on the stock solution of the test substance, serial dilutions were made using solvent to obtain 12 master concentrations to be tested (from 0.098 to 200 mM). The master concentrations were then further diluted in treatment culture medium containing 1% serum, so that the final concentrations of the test substance range from 0.98 to 2000 μM. The solvent DMSO was used as the negative control. Six wells per plate were prepared. It was diluted following the same dilution scheme as described for the master concentrations, so that the final negative control concentration is 1%, which is known to not affect cell viability and corresponds to the same concentration of DMSO found in the test substance and in the positive control. Cinnamic aldehyde was used as the positive control. A series of 5 master concentrations ranging from 0.4 to 6.4 mM was prepared in DMSO and diluted as described for the master concentrations, so that the final concentration of the positive control range from 4 to 64 μM.

Application of the test and control substances:
For each test chemical and positive control substance, one experiment is needed to derive a prediction (positive or negative), consisting of at least two independent repetitions each containing three replicates (i.e. n=6). Each independent repetition was performed on a different day with fresh stock solution of test chemicals and independently harvested cells. Cells may come from the same passage however.
After seeding, cells were grown for 24 hours in the 96-well microtiter plates. The medium was then removed and replaced with fresh culture medium (150 μL culture medium containing 1% serum but without Geneticin to which 50 μL of the diluted test and control substances were added). Three wells per plate were carried out containing no cells to assess background values. The treated plates were then incubated for about 48 hours at 37 ± 1 °C in the presence of 5% CO2. Evaporation of volatile test chemicals and cross-contamination between wells by test substances were avoided by covering the plates with a foil prior to the incubation with the test substances.

Luciferase activity measurements:
After the 48 hour exposure time with the test and control substances, cells were washed with a phosphate buffered saline, and the relevant lysis buffer (One GlowTM Luciferase Assay System) for luminescence readings added to each well for an adequate time at room temperature. Plates with the cell lysate will then be placed in the luminometer (Tecan7 Infinite 200Pro) for reading.

Cytotoxicity assessment:
For the KeratinoSensTM cell viability assay, the medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide) and cells incubated for 4 hours at 37 °C in the presence of 5% CO2. The MTT medium will then be removed and cells were lysed by adding 10% aqueous SDS solution to each well overnight or for up to 3 days at 37 °C. After shaking, the absorption was measured at i.e. 620 nm with a photometer (TecanSunrise Magellan Version 7.2).

Positive control results:

The positive control cinnamic aldehyde was run in both repetitions. Cinnamic aldehyde needs to be positive for a run to be accepted (i.e. induction > 1.5 fold). This was the case in both repetitions. The induction at 64 μM and the EC1.5 for cinnamic aldehyde were also calculated. The targets are:
- the average induction in the two replicates for cinnamic aldehyde at 64 μM should be between 2 and 8,
- the EC1.5 value should be between 7 μM and 30 μM or within two standard deviations of the historical mean value.

At least one of these two numerical criteria must be met in order to accept a repetition. In the experiments performed both criteria were fulfilled in both repetitions. In addition, the EC1.5 value of the positive control of 17.17 μM is within two standard deviations of the historical mean.

Key result

Run / experiment:

other: Luciferase induction: the maximal fold-induction over solvent control

Parameter:

other: I max

Value:

2.71

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 2.71 ± 0.57

Key result

Run / experiment:

other: Conc. for which induction of luciferase activity is above the 1.5

Parameter:

other: EC1.5 (uM)

Value:

24.86

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ± 52.01

Key result

Parameter:

other: IC50 (uM)

Value:

757.09

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ± 155.64

Key result

Parameter:

other: IC30 (uM)

Value:

608.08

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ± 151.96

Other effects / acceptance of results:

The maximal average fold induction of the luciferase activity (Imax) value observed at any concentration of the test substance was 2.71 ± 0.57 and the EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was 24.86 ± 52.01 μM.
The IC50 value was 757.09 ± 155.64 μM and the IC30 value was 608.08 ± 151.96 μM for 50% and 30% reduction of cellular viability, respectively.

The KeratinoSensTM prediction of the test substance is considered positive as the following 4 conditions are all met in 2 of 2 repetitions:
- the Imax of 2.71 is higher than 1.5 fold and statistically significantly different as compared to the negative control (as determined by a two-tailed, unpaired Student’s T-test),
- the cellular viability is higher than 70% at the lowest concentration 62.5 μM with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration),
- the EC1.5 value of 24.86 μM is less than 1000 μM,
- there is an apparent overall dose-response for luciferase induction (Spearman's rank correlation coefficient of p<0.01).

The variability of the luminescence reading for the negative control DMSO must be below 20% in each repetition which consists of 6 wells tested in triplicate. The average coefficients of variation (CV) for the negative control were 12.77% or 9.53% for the first or second repetition, respectively. All quality criteria required ffor the test were fulfilled.

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

Conclusions:

Under the study conditions, the test substance revealed sensitising properties in the ARE-Nrf2 Luciferase 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 442D, in compliance with GLP. Two endpoints were measured: luciferase induction after a 48 h treatment with the test substance and cytotoxicity determined with the MTT assay with the same cell batch and employing the same dilutions of the test substance. DMSO was used as solvent control. For Luciferase induction the maximal fold-induction over solvent control (Imax) and the concentration needed to reach an 1.5 fold induction (EC1.5) were calculated. For cytotoxicity the IC50 and IC30 values were interpolated. The test substance was tested at 12 concentrations in the range from 0.98 to 2000 μM. Cinnamic aldehyde tested at five concentrations from 4–64 μM and was used as the positive control. Two independent repetitions with three parallel technical replicates were run with this same set-up, and one parallel plate was prepared for cytotoxicity determination. For the MTT data the % viability was then calculated for each well in the test plate in relation to average of the six solvent control wells. For the luciferase data the average value of the six solvent control wells was set to 1, and for each well in the test plate the fold induction was calculated in relation to this value. All quality criteria required for the test were fulfilled. Under the study conditions, the maximal average fold induction of the luciferase activity (Imax) value observed at any concentration of the test substance was 2.71 ± 0.57 and the EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was 24.86 ± 52.01 μM. The IC50 value was 757.09 ± 155.64 μM and the IC30 value was 608.08 ± 151.96 μM for 50 and 30% reduction of cellular viability, respectively. Under the stduy conditions, the test substance revealed sensitising properties in the ARE-Nrf2 Luciferase test method (Spruth, 2017).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A study was conducted to evaluate the skin sensitisation potential of the test substance, C11-unsatd. DEA (90.6% active), according to OECD Guideline 442C, in compliance with GLP. In the DPRA in chemico method the reactivity of test substance towards model synthetic peptides cysteine and lysine is quantified. Cysteine and lysine percent peptide depletion values are used to categorise a substance in one of four classes of reactivity. Relative peptide concentration was measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm. The test substance was dissolved at a concentration of 100 mM in acetonitrile/highly purified water 1:1 (v:v). Three reference controls containing only 0.5 mM cysteine or lysine peptide solution and acetonitrile and, in addition the solvent acetonitrile/highly purified water 1:1 (v:v) were also included in the HPLC run sequence. The appropriate reference control for the test substance was used to calculate the percent peptide depletion for the test substance. Each sample was tested in triplicate. Cinnamic aldehyde was used as positive control at a concentration of 100 mM in acetonitrile. Treatment with the positive control substance revealed a cysteine and lysine peptide depletion of 78.34% cysteine and 57.98% lysine. All acceptance criteria of validity were fulfilled for the test. The test substance treated samples revealed a cysteine peptide depletion of 2.56% and lysine peptide depletion of 0.65% (mean peptide depletion of 1.605%) and, hence, well below threshold of 6.38%. Under the study conditions, the test substance revealed a mean cysteine and lysine peptide depletion of 1.605% and hence the test substance is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) in the direct peptide reactivity assay (DPRA) (Rehders, 2017).

A study was conducted to evaluate the skin sensitisation potential of the test substance, C11-unsatd. DEA (90.6% active) according to OECD Guideline 442D, in compliance with GLP. Two endpoints were measured: luciferase induction after a 48 h treatment with the test substance and cytotoxicity determined with the MTT assay with the same cell batch and employing the same dilutions of the test substance. DMSO was used as solvent control. For Luciferase induction the maximal fold-induction over solvent control (Imax) and the concentration needed to reach an 1.5 fold induction (EC1.5) were calculated. For cytotoxicity the IC50 and IC30 values were interpolated. The test substance was tested at 12 concentrations in the range from 0.98 to 2000 μM. Cinnamic aldehyde tested at five concentrations from 4–64 μM and was used as the positive control. Two independent repetitions with three parallel technical replicates were run with this same set-up, and one parallel plate was prepared for cytotoxicity determination. For the MTT data the % viability was then calculated for each well in the test plate in relation to average of the six solvent control wells. For the luciferase data the average value of the six solvent control wells was set to 1, and for each well in the test plate the fold induction was calculated in relation to this value. All quality criteria required for the test were fulfilled. Under the study conditions, the maximal average fold induction of the luciferase activity (Imax) value observed at any concentration of the test substance was 2.71 ± 0.57 and the EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was 24.86 ± 52.01 μM. The IC50 value was 757.09 ± 155.64 μM and the IC30 value was 608.08 ± 151.96 μM for 50 and 30% reduction of cellular viability, respectively. Under the stduy conditions, the test substance revealed sensitising properties in the ARE-Nrf2 Luciferase test method (Spruth, 2017).

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).

 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of the in vitro skin sensitisation studies, the substance does not warrant classification for this endpoint according to EU CLP (EC/1272/2008) criteria.