Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 March - 18 April 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)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
The test material is identical to the registered substance, 'Cell Free Harpin Extract of Harpinαβ produced by fermentation'.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No. of test material: lot# R134-2
- Expiration date of the batch: 17 July 2018
- Appearance: Tan, light brown liquid
- Active components: Harpin-ab proteins 0.4% in aqueous solution
- Storage condition of test material: ≤ -70°C, protected from light
- Treatment of test material prior to testing: The test item was thawed before application.

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany
- Number of animals: Not specified
- Characteristics of donor animals: Not specified
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Fresh eyes were collected from the slaughterhouse on the test day and were transported in HBSS (Hanks' balanced salt solution) containing penicillin/ streptomycin on ice to the laboratories.
- Time interval prior to initiating testing: Eyes were collected and the corneas prepared on the test day.
- Indication of any existing defects or lesions in ocular tissue samples: Any defective eyes and corneas were discarded.
- Indication of any antibiotics used: The HBSS contained penicillin and streptomycin

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µL of the test substance
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
The corneas were incubated for 2 hours prior to the illuminance measurement and for a further 90 minutes prior to measurement of optical density.
Number of animals or in vitro replicates:
3 corneas per treatment group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS :
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. The corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber. 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 (fetal bovine serum) and 2 mM L-glutamine (complete RPMI). The prepared corneas were incubated for one hour at 32 ± 1°C.

QUALITY CHECK OF THE ISOLATED CORNEAS :
Before the corneas were mounted in corneal holders, they were visually examined for defects and any defective cornea were discarded. Only corneas that had an initial luminance reading l >l0/ 1.1651 lux were used for the assay.

NUMBER OF REPLICATES :
Three corneas were used for each treatment group (test substance and positive and negative controls)

NEGATIVE CONTROL USED:
Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative control corneas. The negative controls were treated with physiological saline 0.9% NaCl.

POSITIVE CONTROL USED
The positive controls were treated with ethanol 100%.

APPLICATION DOSE AND EXPOSURE TIME :
750 µL of the test substance or the control substance was introduced into the anterior chamber. After 10 minutes incubation at 32 ± 1°C either the test substance or the control substance was removed.

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE :
- Number of washing steps after exposure period: After the test substance or control substance was removed, the epithelium was washed at least three times with MEM (minimum essential medium, containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red).

POST-EXPOSURE INCUBATION:
Following rinsing of the cornea, an illuminance measurement was performed after 2 hours incubation at 32 ± 1°C. The medium was then removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI and the anterior chamber filled with 1 mL of a 4 mg/mL sodium fluorescein solution. The corneas were then incubated for a further 90 minutes at 32 ± 1°C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Measurement of illuminance
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry at 490 nm (OD490) .
- Others: Each cornea was observed visually and pertinent observations were recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The decision criteria as indicated in the TG were used.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean of three measurements
Value:
-0.46
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
EFFECTS:
None of the corneas treated with the test substance showed opacity of the tissue.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: 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.
- Acceptance criteria met for positive control: The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean.
- Range of historical mean in vitro irritation score values for positive control: Mean value (MV) - 2xSD = 29.20; MV + 2xSD = 67.94

Any other information on results incl. tables

Table 1: In vitro irritation score

Cornea No.

Test item

Corrected opacity

Corrected OD490 value

IVIS

1

Negative control

0.50

0.022

1.18

2

0.75

0.045

3

1.05

0.015

MV

0.77

0.027

4

Positive control

31.03

0.958

42.99

5

26.87

1.139

6

26.45

0.878

MV

28.12

0.991

7

Test item

-0.04

-0.007

-0.46

8

-0.33

-0.008

9

-0.52

-0.016

MV

-0.30

-0.011

MV = mean value

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the mean in vitro irritation score of -0.46, the test substance, Harpin-ab Fermentation Extract is not considered to be an eye irritant.