Amaryllidaceae, Allium Cepa L. (fresh bulb), Rutaceae, Citrus Limon (L.) Burm. F. (fresh pulp), extract, sodium chloride

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19/04/2016-21/04/2016

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

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. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Constituent 2

Reference substance name:

Ethanol

EC Number:

200-578-6

EC Name:

Ethanol

Cas Number:

64-17-5

Molecular formula:

C2H6O

IUPAC Name:

ethanol

Constituent 3

Reference substance name:

Amaryllidaceae, Allium Cepa L. (fresh bulb), Rutaceae, Citrus Limon (L.) Burm. F. (fresh pulp), extract, sodium chloride

EC Number:

944-860-3

IUPAC Name:

Amaryllidaceae, Allium Cepa L. (fresh bulb), Rutaceae, Citrus Limon (L.) Burm. F. (fresh pulp), extract, sodium chloride

Test material form:

liquid

Specific details on test material used for the study:

Test item
Description: Yellow liquid
Storage condition: At room temperature
Specific test item requirements
(handling conditions): None
Purity: Not applicable, mixture
Correction factor: No correction factor
Re-test date: 09 February 2017
Disposal of the test item: Destruction (except the archived test item sample) (any remaining test item is kept for at least 6 months after last use in the project and then disposed of according to instructions described in CiToxLAB France in-house procedures)
The test item does not contain surfactants.
Data relating to the characterization of the test item are documented in a test item information sheet (archived with the study files) and a Certificate of Analysis provided by the Sponsor.
The description of the test item was confirmed by the Sponsor in an email dated 04 April 2016 (archived with the study files).
Confirmation of the identity of the test item is the responsibility of the Sponsor.

As the test item is a non-surfactant containing liquid, it was tested undiluted (i.e. in its original form). The test item was a yellow to brownish liquid.
According to CiToxLAB France in-house procedures, the test item was sampled and stored at room temperature until use.

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Origin: bovine eyes obtained from freshly slaughtered cattle at the abattoir EVA, Saint-Pierre-sur-Dives, France.
Age of the cattle: up to 12 months old.
Reason for choice: recommended by regulatory authorities. They are adapted for the evaluation of potential ocular irritants since they are part of the target organ.
Transport from Supplier to CiToxLAB France: the eyes were transported to CiToxLAB France at ambient temperature, immerged in buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)]. A container with smooth internal surfaces was used for the transport to avoid damage to the corneas

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

yes, concurrent positive control

Amount / concentration applied:

Applied undiluted.
As the test item was a non viscous liquid and could be sampled using a micropipette, a volume of 750 μL (± 8 μL) was applied on each cornea using the closed-chamber method as follows: the test item was introduced into the anterior chamber of the corneal holder, through the dosing holes to cover the epithelial side of the cornea. The dosing holes were then sealed.

Duration of treatment / exposure:

10 minutes (± 30 seconds) (because the test item is a non surfacant liquid)

Duration of post- treatment incubation (in vitro):

Following the 10-minute treatment, the holders were incubated horizontally (corneas placed vertically) for 2 hours (± 10 minutes) in a water bath at +32°C (± 1°C)

Number of animals or in vitro replicates:

3 replicates for each serie (positive control, negative control, test item)

Details on study design:

Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C.
A single experiment was performed using three corneas for each treated series (test item, positive control and negative control).
Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer. The test item, applied undiluted, and the positive and negative controls were evaluated in a single experiment using a treatment time of 10 minutes and using the closed-chamber method. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed.
The corneas were then incubated for 2 hours (± 10 minutes) at +32°C before a second opacity measurement was performed.
After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes (± 5 minutes) at +32°C. At the end of the incubation period, the optical density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

ca. 22

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

For the validation of an experiment, the following criteria had to be fulfilled:
- the mean In Vitro Irritancy Score (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.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Executive summary:

The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 22. As the test item-induced a mean IVIS > 3 and ≤ 55, the eye hazard potential of the test item could not be predicted.

Under the experimental conditions of this study, the ocular corrosive or severe irritant potential of the test item, Extrait de plantes KEOC Version 3, could not be predicted. The test item could not be identified as inducing serious eye damage (UN GHS Category 1) or as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category). According to OECD Guideline 437, further testing should be conducted for classification and labeling purposes.