Registration Dossier

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:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
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. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Name: RCX 15-116
Batch No.: 15047AE2
Purity: > 99%
CAS No.: 15305-07-4
Chemical Name: Aluminium-N-nitrosophenylhydroxylamine (NPAL)
Aggregate State at RT: solid
Colour: whitish
Storage Conditions: room temperature, protected from light
Stability: instable after repeated contact to air and / or light, undergoes hydrolysis
Expiry Date: 30 Nov 2016
Safety Precautions: the routine hygienic procedures were sufficient to assure personnel health and safety.

Test animals / tissue source

Species:
other: cows

Test system

Vehicle:
physiological saline
Amount / concentration applied:
750 µL of the pure test item preparation or the control substance was applied.
Duration of treatment / exposure:
fter 4 hours ± 5 minutes incubation at 32 ± 1 °C 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. Also, each cornea was observed visually, no 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 5 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 (Jenway 6405 UV/VIS).
Number of animals or in vitro replicates:
3 corneas for the test item, 3 corneas as negative controls treated with physiological saline 0.9% NaCl, 3 corneas as positive controls treated with imidazole 20% in physiological saline 0.9% NaCl.

The BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean. The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.
Details on study design:
Preparation of the Corneas
The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany. On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated. The eyes were carefully examined for defects and any defective eyes were discarded. 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. Before the corneas were mounted in corneal holders (Duratec GmbH) 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. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C.

Calibration of the Opacitometer
The opacitometer (BASF-OP3.0, Duratec GmbH) was switched on at least 15 min before starting the calibration procedure. The filter holder was placed into the opacitometer and the readout was adjusted to 1000 lux ± 10 lux using the “Calibrate”-turning knob. For calibration the glass filter F2 was introduced into the filter holder. The readout lied in the range between 540-560 lux. To test the linearity of the measurement, two additional calibration filters, glass filter F3 and glass filter F4, were measured. For these glass filters, the opacitometer displayed values between 300-310 lux and between 95-105 lux. The calibration procedure was performed before the test and is documented in the raw data.

Treatment of the Corneas
After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI. An initial measurement was performed on each of the corneas using the 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. 750 µL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). After 4 hours ± 5 minutes incubation at 32 ± 1 °C 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. Also, each cornea was observed visually, no 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 5 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 (Jenway 6405 UV/VIS).

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Value:
0.63
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Value:
1.02
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Irritation parameter:
other: permeability
Value:
0.026
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
RCX 15-116 did not show any irritancy potential in the bovine corneal opacity and permeability assay.
Executive summary:

The eye irritancy potential of RCX 15-116 was investigated in the bovine corneal opacity and permeability assay. Technically not possible to gain a 20% concentration, therefore the test item was applied directly.

The following mean in vitro irritation score was calculated: 1.02. Therefore the test item does not need to be classified as an eye irritant.

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