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Toxicological information

Skin sensitisation

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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

Constituent 1
Chemical structure
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

Resultsopen allclose all
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
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
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
CV70 [442E]
Value:
18.5 µg/mL
Vehicle controls validity:
valid
Positive controls validity:
valid
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).