Tribenzylamine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-12-01 to 2020-12-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

U937 cell line activation test (U-SENS™)

Test material

Test material information

Constituent 1

Reference substance name:

Tribenzylamine

EC Number:

210-638-3

EC Name:

Tribenzylamine

Cas Number:

620-40-6

Molecular formula:

C21H21N

IUPAC Name:

tribenzylamine

Test material form:

solid: particulate/powder

In vitro test system

Details of test system:

U-937 cell line [442E]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: On the day of the experiment, the test item was dissolved in DMSO to prepare a stock solution with a concentration of 50 mg/mL.
- Preparation of the test chemical serial dilutions: Dilutions in DMSO were prepared from the stock solution and further dilutes with culture medium. By mixing the dilutions with the cell suspension, the final treatment concentrations were achieved.
- Preparation of the positive control: in culture medium
- Preparation of the solvent: DMSO was used; medium control: culture medium was used; and negative control: in culture medium
- Stable dispersion obtained: yes
- Log Kow of the test chemical: > 6.5

DOSE RANGE FINDING ASSAY:
No dose range finder conducted. Doses were adjusted for experiment 2 based results from experiment 1. The highest test item concentration was 200 µg/mL in accordance to the OECD Guideline 442E.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: The dilutions of the test item were tested in two replicates (one labelled with anti-CD86 and one with mouse IgG1). The control groups were tested in six replicates (three labelled with anti-CD86 and three with mouse IgG1).
- Number of repetitions: 2 independent experiments
- Test chemical concentrations: experiment 1: 1, 10, 20, 50, 100 and 200 µg/mL; experiment 2: 1, 5, 10, 15, 20 and 30 µg/mL
- Application procedure: Each volume (100 µL) of the dilutions of the test item, culture medium, positive, negative and solvent control were added to the cells according to the plate template.
- Exposure time: 45 ± 3 h
- Study evaluation and decision criteria used:
For each viable condition (“%viability mean” ≥ 70%), the % of IgG1 positive cells were subtracted to the % of CD86 positive cells. The results were expressed as stimulation index (S.I.) and calculated as S.I.= ((% of CD86 treated cells - % of IgG1 treated cells)/(% of CD86 control cells - % of IgG1 control cells))x100.
The percentage of control cells (solvent/vehicle, i.e. complete medium or DMSO) was the mean of the 3 values obtained, unless one (outlier) was clearly out of the range of the other two.
Cell viability=(Number of living cells/ Total number of acquired cells)x100
If possible, the CV70 value and the EC150 value were calculated in the U-SENS™ test method by log-linear interpolation using the following equitation:
CV70 = C1+[(V1-70)/(V1-V2)*(C2-C1)]
Where:
V1: is the minimum value of cell viability over 70 %
V2: is the maximum value of cell viability below 70 %
C1 and C2 are the concentrations showing the value of cell viability V1 and V2 respectively

EC150 = C1+[(150-S.I.1)/(S.I.2-S.I.1)*(C2-C1)]
Where:
C1 is the highest concentration in µg/mL with a CD86 S.I. < 150 % (S.I.1)
C2 is the lowest concentration in µg/mL with a CD86 S.I. ≥ 150 % (S.I.2)

- Description on study acceptance criteria:
The following acceptance criteria should be met when using the U-SENS™ method:
At the end of the exposure period, the mean viability of the triplicate untreated U937 cells should be more than 90 % and no drift in CD86 expression is observed. The CD86 basal expression of untreated U937 cells had to be comprised within the range of ≥ 2 % and ≤ 25 %.
When DMSO is used as a solvent, the validity of the DMSO vehicle control is assessed by calculating a DMSO S.I. compared to untreated cells, and the mean viability of the triplicate cells had to be > 90 %. The DMSO vehicle control is valid if the mean value of its triplicate CD86 S.I. was smaller than 250 % of the mean of the triplicate CD86 S.I. of untreated U937 cells.
The runs are considered valid if at least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6 % and < 1.5 %.
The negative control is considered valid if at least two out of the three replicates were negative (CD86 S.I. < 150 %) and non-cytotoxic (cell viability ≥ 70 %).
The positive control is considered valid if at least two out of the three replicates were positive (CD86 S.I. ≥ 150 %) and non-cytotoxic (cell viability ≥ 70 %).

SEEDING AND INCUBATION
- Seeding conditions: On the day of the experiment (U-SENS™) directly before the treatment of the cells, a volume of 100 µL with a cell density of 5  10^4 U937 cells/mL was seeded in each corresponding well of a 96-well flat bottom plate. Cells were collected in passage 8 for the first experiment and 10 for the second experiment.
- Incubation conditions: 37 ± 1.5 °C and 5.0 ± 0.5 % carbon dioxide atmosphere
- Washing conditions: washed with approx. 100 µL of staining buffer (PBS with 5 % (w/v) FBS)

MEASUREMENT OF CELL SURFACE EXPRESSION/LUCIFERASE ACTIVITY
- Flow cytometry used: yes. flow cytometer: FACSCalibur, Becton Dickinson GmbH; using the software Cellquest Pro 6.0
- Plate used: For measurement samples were in microtubes and each was placed in a proper cytometer tube.
- cytotoxicity measurements: using Geometric mean fluorescence intensity (GeoMean(7-AAD)) for cytotoxicity (7-ADD=7-amino-actinomycin D)
- Preparation for CD54 and/or CD86 expression measurements/cell staining: All cells were transferred according to the plate template in a v-shape 96-well plate, collected by centrifugation (approx. 200  g, 5 min) and then washed with approx. 100 µL of staining buffer (PBS with 5 % (w/v) FBS). Thereafter, the cells were centrifuged, and the cell pellets were re-suspended in 100 µL staining buffer. The cells were stained with FITC-labelled anti-CD86 and mouse IgG1 (isotype control) and incubated light protected for 30 ± 5 min. on ice.

DATA EVALUATION
- Cytotoxicity assessment: see above
- Prediction model used: The Test Item was tested in at least four concentrations and in at least two independent runs to derive as single prediction (CD86 NEGATIVE or CD86 POSITIVE).
The individual conclusion of an U-SENS™ was considered NEGATIV (N) if the S.I. of CD86 is less than 150 % at all non-cytotoxic concentrations (cell viability ≥ 70 %) and if no interference (defined as IgG1 FL1 Geo Mean S.I. ≥ 150 %) was observed.
In all other cases: S.I. of CD86 ≥ 150 % or interference is observed, the individual conclusion of an U-SENS™ run was considered POSITIVE (P).
An U-SENS™ prediction will be considered negative if the first two independent runs are negative, a third run is not necessary.
An U-SENS™ prediction will be considered positive if the first two independent runs are positive, a third run is not necessary.
Because a dose finding assay is not conducted, there is an exception if, in the first run, the S.I. of CD86 is ≥ 150% at the highest non-cytotoxic concentration only. The run will be considered as not conclusive (NC), and additional concentrations should be tested in additional runs.
In case a run will be identified as not conclusive, at least two additional runs should be conducted, and a fourth run in case runs 2 and 3 are not concordant. Follow up runs will be considered positive even if only one non-cytotoxic concentration gives a CD86 ≥ 150 %, since the concentration setting has been adjusted for the Test Item. The final prediction will be based on the majority result of the four or four individual runs.

Vehicle / solvent control:

DMSO

Negative control:

DL-Lactic acid

Positive control:

picrylsulfonic acid/2,4,6-trinitro-benzene-sulfonic acid (TNBS) [442E]

Results and discussion

Positive control results:

Please refer to "Any other information on results".

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC150, CD86 [442E]

Value:

5.9 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 2

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC150, CD86 [442E]

Value:

1.5 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 3

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

CV70 [442E]

Value:

18.5 µg/mL

Vehicle controls validity:

valid

Positive controls validity:

valid

Results 4

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

CV70 [442E]

Value:

26 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: The technical proficiency of the U-SENS™ with the OECD 442E guideline recommended proficiency substances was demonstrated.

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

Any other information on results incl. tables

Table 1: Results of the first run

		Microscopic evaluation
Test Group	Concen-tration [µg/mL]	Precipitation	Cytotoxicity	MEAN cell viability (%)	S.I. CD86 -IgG1 (%)	Interference IgG1 FL1 Geo Mean S.I. (%)
Medium control		no	no	97.2	93.5	92.9
	/			97.3	87.2	95.8
				97.5	119.3	111.3
	Mean			97.3	100.0	100.0
Negative control		no	no	97.6	132.3	95.9
	200			97.4	132.7	97.4
				97.7	113.5	93.8
Positive control		no	no	97.4	272.0S	108.8
	50			97.7	273.3S	106.1
				97.1	262.0S	97.4
DMSO		no	no	98.0	103.5	89.7
	/			97.3	128.1	87.7
				97.8	91.4	87.9
	Mean					88.4
Test Item	1	no	no	97.7	96.5	94.3
	10	no	no	88.2	1084.5S	108.6
	20	no	no	83.9	491.9S	124.5
	50C	no	yes	14.1	2687.6S	186.1S
	100PC	yes	yes	14.8	2602.3S	191.4S
	200PC	yes	yes	64.0	835.3S	117.9
Outcome:		positive
Calculated EC150:		1.5 µg/mL
Calculated CV70:		26.0 µg/mL

C= test groups with cytotoxic effects (cell viability < 70%) were excluded from the assessment.

S= with S.I. values of ≥ 150% .

P= Precipitations were observed

 

Table 2: Results of the second run

		Microscopic evaluation
Test Group	Concen-tration [µg/mL]	Precipitation	Cytotoxicity	MEAN cell viability (%)	S.I. CD86 -IgG1 (%)	Interference IgG1 FL1 Geo Mean S.I. (%)
Medium control		no	no	97.0	100.4	98.5
	/			97.1	124.4	102.6
				97.2	75.2	98.9
	Mean			97.1	100.0	100.0
Negative control		no	no	97.4	65.4	102.6
	200			97.0	72.9	103.1
				97.5	56.2	97.6
Positive control		no	no	96.6	263.4S	108.1
	50			96.9	294.9S	104.6
				96.6	200.5S	100.7
DMSO		no	no	97.6	73.6	93.5
	/			97.5	139.3	95.2
				97.0	72.0	84.7
	Mean			97.4	94.9	91.1
Test Item	1	no	no	97.4	113.4	101.6
	5	no	no	96.9	133.7	110.4
	10	no	no	95.9	227.8S	121.4
	15	no	no	89.4	488.6S	125.5
	20C	no	no	62.0	931.9S	143.2
	30C	no	no	5.1	2533.7S	286.9S
Outcome:		positive
Calculated EC150:		5.9 µg/mL
Calculated CV70:		18.5 µg/mL

C= test groups with cytotoxic effects (cell viability < 70%) were excluded from the assessment.

S= with S.I. values of ≥ 150% .

 

Table 3: Historical Control Data

		Min	Max	Mean	SD	n
Medium control	Mean relative viability [%]	95	98.41	124.4	0.74	48
	S.I.	75.2	124.4	100.04	10.09	48
Negative control (Lactic acid 200µg/mL)	Mean relative viability [%]	95.8	98.2	97.36	0.67	48
	S.I.	51	148	88.85	25.61	48
Positive control TNBS 50µg/mL)	Mean relative viability [%]	94.8	98.2	97.03	0.84	48
	S.I.	152	446	256.72	66.07	48
Vehicle control	Mean relative viability [%]	94.5	98.2	97.48	0.72	48
	S.I.	63	167	103.95	26.94	48

 

Applicant's summary and conclusion

Interpretation of results:

other: positive for the third key event of the skin sensitisation Adverse Outcome Pathway

Conclusions:

The test item activated U-937 cells under the test conditions of this study. Therefore, the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Executive summary:

An in vitro skin sensitization test (U-SENS™) according to OECD TG 442E was performed to assess the skin sensitization potential of the test item dissolved in DMSO when administered to U937 cells for 45 ± 3 hours. This human cell line activation test can be used as part of a testing battery (including e.g. DPRA (Direct Peptide Reactivity Assay), and ARE-Nrf2 (luciferase test method)) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals. The highest test item concentration was 200 μg/mL in accordance to the OECD TG 442E. For CD86 expression measurement the following concentrations of the test item were tested in the main experiments (U-SENS™): 1, 10, 20, 50, 100 and 200 μg/mL in first experiment (run) and 1, 5, 10, 15, 20 and 30 μg/mL in the second experiment. In the first experiment, cytotoxic effects (cell viability < 70 %) were observed following incubation with the test item starting with a concentration of 50 μg/mL up to the highest test concentration of 200 μg/mL, and precipitation was observed at 100 and 200 μg/mL. Therefore, the concentrations from 50 μg/mL up to 200 μg/mL were excluded from the assessment. In the second experiment cytotoxic effects were observed in the flow cytometric evaluation following incubation with the test item starting with 20 μg/mL up to the highest test concentration of 30 μg/mL. Therefore, the concentrations 20 μg/mL and 30 μg/mL were excluded from the assessment. The CV70 value of the test item was 26.0 μg/ml in the first experiment and 18.5 μg/mL in the second experiment. The test item showed interference (defined as IgG1 FL1 Geo Mean S.I. ≥ 150 %) at 50 μg/mL and 100 μg/mL in the first experiment and at 30 μg/mL in the second experiment. This observation has no impact for the outcome of the study, since the concentrations which showed interference did not meet the cytotoxcitiy criterion and were excluded from the assessment. In the first experiment the CD86 stimulation index (S.I.) was higher or equal to 150 % after treatment with the test item between 10 μg/mL and the highest tested concentration of 200 μg/mL. Two of these concentrations (10 and 20 μg/mL) were non-cytotoxic (cell viability ≥ 70 %). In the second experiment the CD86 stimulation index (S.I.) was higher or equal to 150 % after treatment with the test item between 10 μg/mL and the highest tested concentration of 30 μg/mL. Two of these concentrations (10 and 15 μg/mL) were non-cytotoxic (cell viability ≥ 70 %). Therefore, the outcome of the U-SENS™ is considered positive for the test item. In conclusion, the test item activated U-937 cells under the test conditions of this study. Therefore, the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).