4-[2-[4-[benzylmethyl(ethyl)amino]phenyl]vinyl]-1-(2-hydroxyethyl)pyridinium acetate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-[2-[4-[benzylmethyl(ethyl)amino]phenyl]vinyl]-1-(2-hydroxyethyl)pyridinium acetate

EC Number:

281-468-5

EC Name:

4-[2-[4-[benzylmethyl(ethyl)amino]phenyl]vinyl]-1-(2-hydroxyethyl)pyridinium acetate

Cas Number:

83950-14-5

Molecular formula:

C24H27N2O.C2H3O2

IUPAC Name:

4-[2-[4-[benzylmethyl(ethyl)amino]phenyl]vinyl]-1-(2-hydroxyethyl)pyridinium acetate

Test material form:

liquid

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany (no details)
- Storage, temperature and transport conditions of ocular tissue: transported in HBSS containing Pen/Strep on ice, stored for < 30 min in HBBS
- Time interval prior to initiating testing: incubated in corneal holders in RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine for 1 hour at 32 ± 1 °C
- indication of any existing defects or lesions in ocular tissue samples: any corneas with defects were discarded
- Indication of any antibiotics used: yes see above

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 uL in the anterior chamber of the holder (applied directly onto the cornea due to high viscosity)

Duration of treatment / exposure:

10 min at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

After 10 minutes incubation either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed after 2 hours incubation at 32 ± 1 °C .

Number of animals or in vitro replicates:

3 for test substance, 3 for negative controls and 3 for positive controls

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: from slaughter house animals
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS for <30 min. Before the corneas were mounted in corneal holders with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded.

QUALITY CHECK OF THE ISOLATED CORNEAS: at arrival in the laboratory, before use and after exposure visually inspection for defects

NUMBER OF REPLICATES: 3/group

NEGATIVE CONTROL USED: physiological saline

POSITIVE CONTROL USED: ethanol

APPLICATION DOSE AND EXPOSURE TIME: 750 uL during 10 min at 32 ± 1 °C

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes 2 hours at 32 ± 1 °C

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: washed at least three times with MEM (containing phenol red). The washing medium was thereafter free of coloring, but the corneas showed a strong reddish discoloration..
- POST-EXPOSURE INCUBATION: 2 hours at 32 ± 1 °C

METHODS FOR MEASURED ENDPOINTS:
Before exposure: An initial measurement was performed on each of the corneas using an opacitometer. Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay. The medium was removed from the anterior chamber and replaced with the test item or control.

After exposure: Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed after 2 hours incubation at 32 ± 1 °C. Also, each cornea was observed visually and pertinent observations were recorded.
After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 4 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer

- Corneal opacity: BASF opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured at 490 nm (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: according to the guidance
IVIS UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

141.46

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

the tissue treated with the substance showed strong reddish discolouration and therefore it cannot be excluded that these have interfered with the opacity readings

Any other information on results incl. tables

In Vitro Irritation Score

Group	Cornea no.	Corrected opacity	Corrected OD490	IVIS
Negative control	1	-0.25	0.004
	2	0.80	0.005
	3	1.17	0.008
	Mean value	0.57	0.006	0.66
Positive control	1	23.92	1.436
	2	31.18	1.102
	3	29.69	1.749
	Mean value	28.26	1.429	49.70
Test substance	1	153.43	0.344
	2	127.34	0.369
	3	129.19	0.308
	Mean value	136.65	0.341	141.76

 

historical controls

	IVIS positive control ethanol
Mean value	48.40
SD	9.98
Mean value + 2*SD	28.44
Mean value – 2*SD	68.37
Based on 45 replicates

 

 

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Based on the results of the test the substance is considered to be severely irritant to the eyes

Executive summary:

The eye irritancy potential of the substance was investigated in the bovine corneal opacity and permeability assay.

All 3 corneas treated with the substanceshowed strong reddish discolouration of the tissue.

The following mean in vitro irritation score was calculated: 141.76

The in vitro score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.