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Eye irritation

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eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Nov 2017 - 17 Nov 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to 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:
Adopted October 09, 2017
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
(6S)-6-(propylamino)-5,6,7,8-tetrahydronaphthalen-1-ol hydrobromide
EC Number:
Cas Number:
Molecular formula:
(6S)-6-(propylamino)-5,6,7,8-tetrahydronaphthalen-1-ol hydrobromide
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: White powder
- Storage condition of test material: At room temperature

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
- Amount(s) applied: 52.7 and 58.3 mg

- Amount(s) applied: 50 µL
Duration of treatment / exposure:
6 hours
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
Details on study design:
- Details of the test procedure used : The test substance was applied directly on the Reconstructed Human EpiOcular™ Model. The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo. After 6 hours exposure, the tissue construct is thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 18 hours at standard culture conditions, prior todetermination of the cytotoxic (irritancy) effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment.
- RhCE tissue construct used, including batch number : EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 27406 Kit Q
- Duration and temperature of exposure period: 6 hours ± 15 minutes at 37.0 ± 1.0°C
- Duration and temperature of post-exposure immersion period: 25 ± 2 minutes at room temperature
- Duration and temperature of post-exposure incubation period: 18 hours ± 15 minutes at 37°C
- Number of tissue replicates used per test chemical and controls: 2
- Description of the method used to quantify MTT formazan : The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model : Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item. The test item is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60% compared to the negative control tissues. The test item is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and postexposure incubation is less than or equal (≤) to 60% compared to the negative control tissues.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria :
Negative control Positive control Positive control
(absorption; OD570) (absorption; OD570) (viability; %)
Range 1.228 – 2.090 0.320 – 0.535 17.80 – 34.18
Mean 1.53 0.42 27.55
SD 0.23 0.07 5.31
n 12 12 12
SD = Standard deviation
n = Number of observations
The above mentioned historical control data range of the controls were obtained by collecting all data over the period of January 2013 to May 2017.
- Acceptability criteria (for test substances not inducing color interference and not interacting with MTT):
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and < 2.5
b) The mean relative tissue viability of the positive control should be <50% relative to the negative control
c) The difference between the % tissue viabilities of two identically treated replicates should be <20

Results and discussion

In vitro

Irritation parameter:
in vitro irritation score
Run / experiment:
Mean of 2 replicates
Negative controls validity:
Positive controls validity:
Other effects / acceptance of results:
- Acceptance criteria met for negative control: The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range (and > 0.8 and < 2.5)
- Acceptance criteria met for positive control: The positive control had a mean cell viability after 6 hours ± 15 minutes exposure of <50% (31%)
- The difference between the % tissue viabilities of two identically treated replicates was <20 (<11)

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Using the EpiOcular™ cornea epithelial model in accordance with OECD 492, it was determined that the substance does not need to be classified for eye irritation/corrosion.
Executive summary:

Using the EpiOcular™ cornea epithelial model the substance was screened for its eye irritancy potential in accordance with OECD 492 and according to GLP principles. An amount of 52.7 to 58.3 mg of the test substance was applied directly on top of the tissue for 6 hours in duplicate. Adequate negative and positive controls were included. Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 6 hours treatment with the test substance compared to the negative control tissues was 65%. The acceptability criteria were met. Since the mean relative tissue viability for the test substance was above 60%, the substance is considered to be non-irritant. The substance does not need to be classified for eye irritation/corrosion in accordance with GHS/CLP.