(6S)-6-{propyl[2-(2-thienyl)ethyl]amino}-5,6,7,8-tetrahydronaphthalen-1-ol hydrochloride (1:1)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Jan 2018 - 04 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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:

October 09, 2017

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(6S)-6-{propyl[2-(thiophen-2-yl)ethyl]amino}-5,6,7,8-tetrahydronaphthalen-1-ol hydrochloride

EC Number:

603-080-0

Cas Number:

125572-93-2

Molecular formula:

C19H25NOS*HCl

IUPAC Name:

(6S)-6-{propyl[2-(thiophen-2-yl)ethyl]amino}-5,6,7,8-tetrahydronaphthalen-1-ol hydrochloride

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: White powder
- Storage condition of test material: At room temperature

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amounts applied: 60.7 to 67.3 mg

CONTROLS
- Amount 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:

2

Details on study design:

- Details of the test procedure used : The test substance was topically applied 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 to determination 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: 27427 Kit A)
- 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

As it was determined that the test substance did not induce color interference in aqueous conditions, but did interact with the MTT endpoint, the following tissues were tested:
- Number of tissue replicates used per test chemical: 2
- Number of tissue replicates used per test chemical (NSCkilled): 2
- Number of tissue replicates used per test controls (positive control and negative control): 2
- Number of tissue replicates used per test control (NSCkilled): 1

- 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 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
d) The non-specific MTT reduction should be <= 50% relative to the negative control OD.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean of 2 replicates

Value:

45

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

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) (1.747)
- Acceptance criteria met for positive control: The positive control had a mean cell viability after 6 hours ± 15 minutes exposure of <50% (32%)
- The difference between the % tissue viabilities of two identically treated replicates was <20 (<16)
- The non-specific MTT reduction was <= 50% relative to the negative control OD (19%)

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Using the EpiOcular™ cornea epithelial model in accordance with OECD 492, it was determined that the substance is considered to be potentially irritant or corrosive to the eye.

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 60.7 to 67.3 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. As it was determined that the test substance did directly interact with MTT, two freeze-killed tissues treated with test item and one freeze-killed negative control treated tissue were additionally used, for the cytotoxicity evaluation with MTT. The ODs of the test item treated viable tissues was corrected using the OD of the freeze-killed tissues. 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 45%. The acceptability criteria were met. Since the mean relative tissue viability for the test substance was below or equal to 60%, the substance is considered to be potentially irritant or corrosive to the eye. Using this study, the substance is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).