Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Jul - 01 Sept 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
/ No demonstration of technical proficiency

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

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:
26 July 2013
Deviations:
yes
Remarks:
/ no demonstration of technical proficiency
GLP compliance:
yes (incl. certificate)
Remarks:
Secrétariat général du GIPC - DGE - SI, Ivry-sur-Seine, France

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light

Handling: under inactinic light

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: abattoir EVA, Saint-Pierre-sur-Dives, France
- Characteristics of donor animals: maximum 12 month old donor cattle (typically 5 to 8 months)
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were transported in HBSS (with streptomycin / penicillin) in a cooling box.
- Time interval prior to initiating testing: Test was initiated as soon as possible after delivery of the eyes.
- indication of any existing defects or lesions in ocular tissue samples: no
- Indication of any antibiotics used: streptomycin / penicillin

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 (± 8) µL
- Concentration (if solution): undiluted
Duration of treatment / exposure:
10 min ± 30 sec (at 32 ± 1 °C)
Duration of post- treatment incubation (in vitro):
2 h ± 10 min (at 32 ± 1 °C)
Number of animals or in vitro replicates:
Number of eyes for the test item/ negative control/ positive control: 3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS: A careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded. The tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared.

QUALITY CHECK OF THE ISOLATED CORNEAS: yes, eyes were free of defects

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: yes

POSITIVE CONTROL USED: yes

APPLICATION DOSE AND EXPOSURE TIME: 750 µl test item (100%), 10 min

TREATMENT METHOD: open chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 6. The corneas were rinsed 5 times with pre-warmed cMEM containing phenol red. Then, they were finally rinsed with pre-warmed cMEM without phenol red.

- POST-EXPOSURE INCUBATION: yes, 2 h

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: determined by the amount of light transmission through the cornea via an opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of spectrophotometry (OD490).
- Others: After permeability determination, the corneas were removed from the holders and observed for opaque spots, other irregularities and any separation of the epithelium (macroscopic examination).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The decision criteria as indicated in OECD 437 was used.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
positive control - mean out of all 3 eyes
Value:
20.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
test item - mean out of all 3 eyes
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein leakage
Remarks:
permeability
Run / experiment:
positive control - mean out of all 3 eyes
Value:
1.151
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein leakage
Remarks:
permeability
Run / experiment:
test item - mean out of all 3 eyes
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
positive control - mean out of all 3 eyes
Value:
38
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
test item - mean out of all 3 eyes
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: Application of the test item did not induce corneal opacity or altered permeability. Based on the estimated scores, an IVIS of 1 was determined. Fluorescein fixation was observed on the corneas treated with the test item. Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.

DEMONSTRATION OF TECHNICAL PROFICIENCY: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Acceptance criteria
For the validation of an experiment, the following criteria had to be fulfilled:
- the mean IVIS of the positive control corneas should fall within two standard deviations of the historical mean
- the mean opacity of the negative control corneas should be < 1.8
- the mean OD490 nm of the negative control corneas should be < 0.0269

Any other information on results incl. tables

Table 1: Opacity values

Parameter

Initial opacity (OPT0)

Final opacity (OPT2)

Opacity change (OPT2-OPT0)

Mean opacity change of NC (after negative correction)

Corrected opacity change

(cOPT)

Mean opacity value

Negative control

0

-1

-1

0

-

-

0

0

0

0

0

0

Test substance

-2

0

2

-

2

1.0

1

1

0

0

1

2

1

1

Positive control

-1

21

22

-

22

20.2

0

22

22

22

2

19

17

17

 

Table 2: Permeability values (optical density (OD) at 490 nm)

Parameter

OD490 change

Mean OD490 change of NC

Corrected OD490 change

Mean OD490 value

Negative control

0.008

0.009

-

-

0.009

0.009

Test substance

0.000

-

0.000

0.000

0.000

0.000

0.000

0.000

Positive control

1.392

-

1.383

1.151

1.312

1.303

0.774

0.765

 

Table 3: In-Vitro Irritancy Score (IVIS) values

 

IVIS

Mean IVIS

Test substance

2

1

0

1

Positive control

43

38

42

28

 

Applicant's summary and conclusion

Interpretation of results:
other: CLP/ EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
CLP: not classified