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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Nov 2017 - 09 Jan 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:
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)
Version / remarks:
Adopted 09 Oct 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
GLP-Landesleitstelle Bayern, Bayerrisches Landesamt für Gesundheit und Lebensmittelsicherheit.

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany.
- Number of ocular tissues: 9 corneas. 3 for the test item, 3 for negative controls and 3 for positive controls.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Fresh eyes transported in HBSS containing Pen/Strep on ice to the laboratories; the isolated corneas mounted in corneal holders (Duratec GmbH) which filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI) , then the corneas were incubated for one hour at 32 ± 1 °C.
- Time interval prior to initiating testing: 1 hour
- indication of any existing defects or lesions in ocular tissue samples: visually examined for defects.

Test system

Vehicle:
physiological saline
Remarks:
0.9% NaCl
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 750 μL/cornea
Duration of treatment / exposure:
4 h ± 5 min
Duration of post- treatment incubation (in vitro):
90 minutes
Number of animals or in vitro replicates:
3 corneas each for the test item, negative and positive controls
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The eyes were carefully examined for defects after arrival of the eyes, any defective eyes were discarded. The tissue surrounding the eyeball was 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.

QUALITY CHECK OF THE ISOLATED CORNEAS
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.

NUMBER OF REPLICATES: 3

SOLVENT CONTROL USED: Physiological saline 0.9% NaCl (B. Braun Melsungen, lot no. 17296405)

POSITIVE CONTROL USED: Imidazole 20% (Sigma-Aldrich, lot no. SCBL7984V) in physiological saline 0.9% NaCl

APPLICATION DOSE AND EXPOSURE TIME: 750 μL/cornea for 4 h ± 5 min

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: washed at least three times with MEM (containing phenol red)

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: illuminance measurement
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry ] (OD490)
- Others (e.g, pertinent visual observations, histopathology): pertinent visual observations

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: IVIS ≤3, non – irritant; 3< IVIS ≤55, No prediction can be made; IVIS >55, irritant.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
104.92
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
750 μL test item (undiluted) per cornea reached an in vitro irritation score of 104.92. According to the evaluation criteria, the test substance is considered as eye damaging.
The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore the 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.

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS Category 1 (H318) according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the test, the test substance was shown corrosive potential in the Bovine corneal opacity and permeability (BCOP) test prediction model.