Registration Dossier
Registration Dossier
Diss Factsheets
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 210-638-3 | CAS number: 620-40-6
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data

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:
- 2 021
- 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
- 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
Constituent 1
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 |
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 |
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).
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
