Tribenzylamine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-10-14 to 2020-10-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

31 July 2019

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

June 18, 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Justification for test system used:

The reconstructed human epidermis model in vitro method is an accepted in vitro method to replace animal testing. The human skin RHE™ model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis, and has been validated by the ECVAM in 2008.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model RHE/S/17 (from Episkin/SkinEthic Laboratories, Lyon, France)
- Tissue batch number: 20-RHE-133
- Production date: not specified
- Shipping date: not specified
- Delivery date: not specified
- Date of initiation of testing: 2020-10-14
- Expiry date: 2020-10-19

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: minimum volume of 25 mL DPBS was used
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours (± 5 minutes)
- Spectrophotometer: ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth: not specified
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Acceptance Criteria: OD >0.7; Result: OD 1.3 (CV = 7.7 %)
- Barrier function: Acceptance Criteria: 4.0 h - Morphology: Acceptance Criteria: Number of cell layers > 4, Multi-layered, highly differentiated epidermis consisting of basal, spinous and granulär layers, and a multi-layered Stratum corneum; Result: 6.5 cell layers, Satisfactory
- Contamination: not specified
- Reproducibility: not specified

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The pre-test for direct MTT-reducing capacity of the test item did not result in blue color, i. e. the test item is not a direct MTT reducer. Therefore, no additional control was needed.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive or irritant to skin if the viability is less than or equal to 50 %.
- The test substance is considered to be non-corrosive and non-irritant to skin if the viability is greater than 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 16 mg ± 2 mg per tissue (10 µL of deionised water used to wet tissue before application)

NEGATIVE CONTROL
- Amount applied: 16 µL ± 0.5 µL per tissue

POSITIVE CONTROL
- Amount applied: 16 µL ± 0.5 µL per tissue
- Concentration: 5 %

Duration of treatment / exposure:

42 minutes (± 1 minute)

Duration of post-treatment incubation (if applicable):

42 hours (± 1 hour)

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

76.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

1.6

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: not specified
- Direct-MTT reduction: The pre-test for direct MTT-reducing capacity of the test item did not result in blue color, i. e. the test item is not a direct MTT reducer.
- Colour interference with MTT: The test item has no colorant properties. Therefore, no interference was observed.

DEMONSTRATION OF TECHNICAL PROFICIENCY: not specified

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values: Please refer to "Any other information on results".

Any other information on results incl. tables

Table 1: Study results

Substance	Optical Density (OD)		Viability (%)
Negative Control 1x PBS	1	1.73	99.4
	2	1.812	104.1
	3	1.679	96.5
	mean	1.74	100
	standard deviation (SD)		3.8
Positive Control SDS (5 % aq.)	1	0.027	1.6
	2	0.026	1.5
	3	0.027	1.6
	mean	0.027	1.6
	standard deviation (SD)		6.3
Test item	1	1.261	72.5
	2	1.326	76.2
	3	1.406	80.8
	mean	1.331	76.5
	standard deviation (SD)		5.5

 

Table 2: Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data

	Acceptance Criterion	Result
Negative control OD	≥0.8 and ≤ 3.0	1.679 to 1.812

 

Table 3: Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories

	Acceptance Criterion	Result
Mean OD negative control	≥ 1.2	1.74
Mean viability positive control	< 40 %	1.6 %
SD of group-mean value	≤ 18 %	6.3 % (positive control) 3.8 % (negative control)

 

Table 4: Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory

	Acceptance Criterion	Result
Mean OD negative control	≥ 1.415	1.74
Mean viability positive control	≤ 2.72 %	1.6 %

 

Table 5: Test Item Data Acceptance Criteria

	Acceptance Criterion	Result
SD of group-mean value	≤ 18 %	5.5 %

 

Table 6: Historical Data (of all tests performed in laboratory)

Positive control		Negative control
Mean viability [%]	1.42	Mean adsorption [OD570]	1.937
Standard deviation	0.44	Standard deviation	0.26

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin.

Executive summary:

The objective of the study conducted according to OECD 439 was to investigate the potential of the test item to induce skin irritation in an in vitro human skin model. The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential. Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (DPBS-buffer) or the positive control (5 % aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before application of 16 mg of the solid test item, 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis. All acceptability criteria after treatment with the negative control and the positive control were met. Following treatment with the test item, the tissue viability was 76.5 % and, thus, higher than 50 %, i.e. according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category).