Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-12-01 to 2020-12-03
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: EU Method B.69 (RECONSTRUCTED HUMAN CORNEA-LIKE EPITHELIUM (RhCE) TEST METHOD FOR IDENTIFYING CHEMICALS NOT REQUIRING CLASSIFICATION AND LABELLING FOR EYE IRRITATION OR SERIOUS EYE DAMAGE)
Version / remarks:
31 July 2019
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
June 18, 2019
Deviations:
no
GLP compliance:
yes (incl. QA statement)

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

Test animals / tissue source

Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability: The reconstructed human cornea-like epithelium (RhCE) model is an accepted in vitro method to replace animal testing. The human eye EpiOcular™-model closely mimics the biochemical and physiological properties of the human eye, i.e. the cornea.
- Description of the cell system used, and the mycoplasma status of the cell live: The EpiOcular™-model is a nonkeratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified cells. Long term antibiotic and antimycotic free culture with no contamination was applicable (result: sterile). Therefore, the QA statement from the supplier considered a pass for the the sterility criterion.
- Cell line used, its source, passage number and confluence of cells used for testing: Keratinocyte strain: 4F1188; passage number: not specified; Barrier
function: 12.93 min (within acceptance criteria of supplier)
- RhCE tissue or hCE cell construct used, including batch number: EpiOcular™ Tissue (OCL-200, OCL-212) from MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia, Lot no: 30687

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 mg

Negative and positive control
- Amount applied: 50 µL
Duration of treatment / exposure:
6 hours (± 15 minutes) at 37 °C
Duration of post- treatment incubation (in vitro):
25 minutes (± 2 minutes) at room temperature and 18 hours (± 15 minutes) at 37 °C
Number of animals or in vitro replicates:
2
Details on study design:
- Details of the test procedure used:
The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) 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 eye irritation potential.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals:
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 and the test item has no colorant properties. Therefore, no further control was needed.
- Wavelength used for quantifying MTT formazan: 570 nm
- Description of the method used to quantify MTT formazan: Spectrophotometer (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany)
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
The test item is identified as not requiring classification and labeling according to UN GHS (No Category) if the mean percent tissue viability is more than 60 %. If the mean percent tissue viability is less than or equal 60 %, no prediction can be made.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria:
The results are acceptable if:
The negative control OD is >0.8 and <2.5.
The mean relative viability of the positive control is:
a) 30-minute exposure (treatment of liquid test items): below 50 % of control viability
b) 6-hour exposure (treatment of solid test items): below 50 % of control viability
- Reference to historical data of the RhCE tissue construct: Tissue viability via MTT QC assay, 1 hour, n=3; Acceptance criteria of OD (540-570 nm) [ 1 . 1 - 3 . 0] with a result of 1.899 ± 0.045 for the batch according to supplier
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: not specified
- Acceptable variability between tissue replicates
The difference of viability between the two relating tissues of a single chemical is <20 % in the same run (for positive and negative control tissues and tissues of single chemicals).

Results and discussion

In vitro

Results
Irritation parameter:
mean percent tissue viability 
Value:
106.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100
Positive controls validity:
valid
Remarks:
39.7
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: not specified

DEMONSTRATION OF TECHNICAL PROFICIENCY: not specified

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The negative control OD is >0.8 and <2.5 (1.732 and 1.686).
- Acceptance criteria met for positive control: yes. The mean relative viability of the positive control is below 50 % of the negative control viability (39.7 %).
- Other: The difference of viability between the two relating tissues of a single chemical is <20 % (values between 0.0 % to 5.1%) in the same run (for positive and negative control tissues and tissues of single chemicals).

Any other information on results incl. tables

























































































SubstanceOptical Density (OD)Viability (%)
Negative Control11.732101.3
water21.68698.7
 mean1.709100
 standard deviation (SD)1.84
 Difference between tissue replicates2.6
Positive Control10.67939.7
SDS (5 % aq.)20.67939.7
 mean0.67939.7
 standard deviation (SD)0
 Difference between tissue replicates0
Test item11.782104.3
21.87109.4
mean1.836106.9
standard deviation (SD)3.61
Difference between tissue replicates5.1

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item did not show an eye irritating potential.
Executive summary:

The objective of the study conducted according to OECD 492 was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model. The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) 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 eye irritation potential. Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 6 hours (± 15 minutes). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues. After treatment with the negative control (sterile deionized water) the OD was 1.732 and 1.686 of the two treated tissue, respectively (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 39.7 % (study acceptance criterion: <50 %). Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was 106.9 % and, thus, higher than 60 %, i.e. according to OECD 492 the test item is identified as not requiring classification and labeling according to UN GHS (No Category).