Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 Feb 2018 - 29 Mar 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 guidelineopen allclose all
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:
2017
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
08 May 2017

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Hubert Bahlmann GmbH & Co. Versandschlachterei Spezialmischfutterwerk KG, 49699 Lindern, Germany
- Characteristics of donor animals: age range of 6 to 12 months
- Storage, temperature and transport conditions of ocular tissue: Eyes were completely submerged in Hanks' Balanced Salt Solution (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL
- indication of any existing defects or lesions in ocular tissue samples: Only corneas from eyes free of defects were used.

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 µL
- Concentration (if solution): undiluted

Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
Opacity: None
Corneal permeability: 90 +/- 5 min
Number of animals or in vitro replicates:
3 per condition
Details on study design:
SELECTION AND PREPARATION OF CORNEAS :
Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation. Only corneas from eyes free of defects were used.
The corneas were dissected with a 2 to 3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers. Beginning with the posterior chambers, the chambers were filled to excess with pre-warmed Eagle's Minimum Essential Medium (EMEM), while preventing bubble formation. The corneal holder was equilibrated at 32±1°C for at least one hour.

QUALITY CHECK OF THE ISOLATED CORNEAS
Corneas that had an opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one hour equilibration period had to be discarded.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
0.9% sodium chloride solution

POSITIVE CONTROL USED
1% NaOH solution in highly purified water

APPLICATION DOSE AND EXPOSURE TIME
750µL, 10 minutes

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: only for corneal permeability measurement 90 +/- 5 min

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3 times, Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer resulting in opacity values measured on a continuous scale.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490, Tecan Sunrise visible light)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

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

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
Cornea No. 7
Value:
-1.742
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Cornea No. 8
Value:
0.639
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Cornea No. 9
Value:
-1.487
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:
OTHER EFFECTS:
- Visible damage on test system: None

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values:

NaCl 0.9%: Opacity 0.062 (sd 0.934), Permeability 0.043 (sd 0.073), IVIS 0.579 (sd 1.097)
NaOH 1%: Opacity 72.67 (sd 20.908), Permeability 2.016 (sd 0.415), IVIS 102.903 (21.283)

Applicant's summary and conclusion

Interpretation of results:
other: Not irritant
Remarks:
in accordance with Annex I of the CLP Regulation (1272/2008/EC).
Conclusions:
Based on the results of the in vitro eye irritation test for Ginger CO2-se extract, the test substance does not need to be classified for skin irritation in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The eye irritant properties of Ginger CO2-se extract were examined in a study performed according to OECD Guideline 437, under GLP. Three corneas were used for each treatment group (test item, negative control and positive control). The liquid test item Ginger CO2-se extractwas used undiluted. 0.9% NaCl solutionwas used as the negative control and 1% NaOH in water (highly purified water)as the positive control item. The test and control items were applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder. The exposure time for the test item and the controls was 10 minutes. The optical density (OD) was measured at a wavelength of 490 nm. The acceptance criteria of validity were fulfilled in this test. Following treatment with Ginger CO2-se extract a mean opacity and a mean permeability value of <0.01compared to the negative control were determined. The calculated IVIS was below the cut-off value of 3and consequently the test item is not classified as a severe irritant and is not considered corrosive.