(2S)-5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-aminium (2R)-(2-chlorophenyl)(hydroxy)acetate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 25, 2013 - March 26, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

2009

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(2S)-5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-aminium (2R)-(2-chlorophenyl)(hydroxy)acetate

EC Number:

814-350-4

Cas Number:

1352917-66-8

Molecular formula:

C22 H28 N Cl O4

IUPAC Name:

(2S)-5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-aminium (2R)-(2-chlorophenyl)(hydroxy)acetate

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Off-white powder with lumps
- Storage condition of test material: At room temperature in the dark

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Since no workable suspension in physiological saline could be obtained, the test substance was added as such on top of the corneas
- Amount applied: 304.3 to 313.5 mg per cornea

Duration of treatment / exposure:

4 hours

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
All eyes were carefully examined for defects by holding the eyes submersed in physiological saline. Those exhibiting unacceptable defects, such as opacity, scratches, pigmentation and neovascularization were discarded.
The isolated corneas were stored at 32 ± 1°C in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Invitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)).
The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont, France) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for 1 hour at 32 ± 1°C.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (OP-KIT, MC2, Clermont, France). The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.

NEGATIVE CONTROL USED
750 µL of physiological saline per cornea

POSITIVE CONTROL USED
750 µL of a 25% (w/v) Imidazole solution in physiological saline per cornea

EXPOSURE TIME
4 hours (at 32°C)

TREATMENT METHOD
The medium from the anterior compartment was removed and the control solutions were introduced onto the epithelium of the cornea.
The test substance was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered.
The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control solutions over the entire cornea.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation)

METHODS FOR MEASURED ENDPOINTS
- Corneal opacity: The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numerical opacity value (arbitrary unit) was displayed and recorded. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading.
- Corneal permeability: Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated, after another incubation period of 85 to 86 minutes at 32°C. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader).
- Others: Each cornea was inspected visually for dissimilar opacity patterns.

SCORING SYSTEM
In Vitro Irritancy Score (IVIS) = mean opacity value + (15 x mean OD490 value)

DECISION CRITERIA
- A test substance that induces a mean IVIS >55 is classified with Eye Irr. Cat. 1 in accordance with the CLP Regulation
- A test substance that induces a mean IVIS ≤ 3 is not classified for Eye irritation/corrosion in accordance with the CLP Regulation
- For a test substance that induces a mean IVIS of >3 - ≤55, no prediction on the classification can be made. More information is needed.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean of 3

Value:

10.7

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Summary of opacity, permeability and in vitro scores:

Treatment	Mean opacity	Mean permeability	Mean in vitro irritation score
Negative control	0	0.000	0.0
Positive control	135	2.544	173.2
Test substance	8	0.183	10.7

 

Individual in vitro irritancy scores:

Eye	In vitro Irritancy Score
Negative control
1	-0.1
2	-0.3
3	0.4
Positive control
4	166.4
5	172.3
6	180.8
Test substance
10	10.3
11	11.5
12	11.5

The corneas were clear after the 240 minutes of treatment with the test substance, however several spots were observed on the corneas.

A pH effect of the test substance was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on a mean in vitro irritancy score of 10.7 in a Bovine Corneal Opacity and Permeability test, it is concluded that no prediction can be made whether the substance needs to be classified for eye irritation/corrosion. For this, more information is needed.

Executive summary:

Using the Bovine Corneal Opacity and Permeability test (BCOP test) the substance was screened for its eye irritancy potential in accordance with OECD 437 and according to GLP principles. Since no workable suspension in physiological saline could be obtained, the substance was applied as such directly on top of the corneas in such a way that the cornea was completely covered (304.3 to 313.5 mg per cornea). Adequate negative and positive controls were included. The mean in vitro irritancy score was 10.7 after 240 minutes of treatment. Since the substance induced a mean IVIS >3 and ≤ 55, no prediction can be made whether the substance needs to be classified for eye irritation/corrosion. For this, more information is needed.