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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:
03 January 2014 -- 13 February 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: EU method B.47 (In vitro eye irritation)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD guideline 437 (In vitro eye irritation)
Deviations:
no
GLP compliance:
yes (incl. certificate)

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:
Species: bovine cattle.
Origin: bovine eyes were obtained from EVA, Saint-Pierre-sur-Dives, France.
Age: bovine cattle were up to 12 months old.
Reason for choice: bovine corneas are recommended by regulatory authorities for this type of study. 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.

Upon arrival at CiToxLAB France, the selection and preparation of corneas was performed as soon as possible. At each step of the preparation procedure, care was taken to avoid touching the corneas in order not to damage them.

Selection: 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 examination was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny. Particular attention was paid to the corneas, and the eyes were swiveled in order to observe the fringe areas and any scratches directly under the light.
Preparation of the selected corneas: the tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared.
Washing of the corneas: the corneas were washed for 15 minutes, three times, in HBSS plus penicillin/streptomycin (100 units/100 µg/mL final) at room temperature.
The prepared corneas were stored and used within 24 hours.
(Pre)Incubation T°C: 32°C

Dates of experimental phase: from 12 February 2014 to 13 February 2014

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: in vitro negative and positive controls
Duration of treatment / exposure:
Exposure period of 10 minutes, followed by rinsing
Duration of post- treatment incubation (in vitro):
Opacity measurement:
- before treatment
- after 2-hour incubation period in a water bath at +32°C (± 1°C).
Permeability measurement:
- after 90-min incubation in water (and other procedures), following the 2nd opacity measurement
Number of animals or in vitro replicates:
Triplicate corneas for each tested substance (test item, negative control, positive control)
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Rinsing: the anterior part of the eye was emptied and then rinsed up to six times with pre-warmed cMEM.

NEGATIVE CONTROL:
As the test item was tested in its original form, the negative control was 0.9% NaCl, batch No. 3A077 supplied by CDM Lavoisier.

POSITIVE CONTROL:
10% sodium hydroxide solution (10% NaOH).

SCORING SYSTEM/TOOL
- Opacity:
Using an opacitometer
The average change in opacity during exposure is determined. It is corrected by subtracting the average negative control value from values in positive control and test item.
- Permeability:
Using a spectrophotometer: optical density (OD) at 490 nm wavelength
The optical density is corrected by subtracting the average negative control value from values in positive control and test item.
- Scoring:
In vitro irritancy score (IVIS) = Corrected Opacity + (15 x Corrected OD)

Interpretation: see below

Results and discussion

Results of ex vivo / in vitro study
Irritation parameter:
in vitro irritation score
Run / experiment:
test
Value:
-1
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Macroscopic examinations : No notable opaque spots or irregularities were observed on test item-treated corneas following the treatment.
In Vitro Irritancy Score : The In Vitro Irritancy Score (IVIS) was: -1.
All acceptance criteria were fulfilled. The study was therefore considered as valid.
Other effects:
no

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, the test item was identified as a test chemical not requiring classification for eye irritation or serious eye damage.
Executive summary:

The objective of this study was to evaluate the eye hazard potential of the test item. Indeed, the Bovine Corneal Opacity and Permeability (BCOP) test method can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage.

The study design was based on the OECD guideline No. 437 and was performed in compliance with CiToxLAB France’s standard operating procedures and with the principles of Good Laboratory Practice.

Method

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.

Three corneas were used for each treated series (test item, positive control and negative control).

Before the treatment, a first opacity measurement was performed using an opacitometer.

The test item was tested undiluted (i.e. in its original form) in a single experiment using a treatment time of 10 minutes and using the closed-chamber method. At the completion of the treatment period, the test and control items were removed from the front opening of the anterior chamber and the corneas were rinsed. The corneas were then incubated for 2 hours at +32°C before a second opacity measurement was performed. After the second opacity measurement, the medium of the anterior chamber was removed for each cornea and filled with a fluoresceine solution. The holders were then incubated vertically for 90 minutes at +32°C.

At the end of the incubation, the optical density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Then the cornea was observed for opaque spots and other irregularities.

Results

Macroscopic examinations

No notable opaque spots or irregularities were observed on test item-treated corneas following the treatment.

 

In Vitro Irritancy Score

All acceptance criteria were fulfilled. The study was therefore considered as valid.

The In Vitro Irritancy Score (IVIS) was: -1. As the test item induced an IVIS = 3, the test item was considered as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category). 

Conclusion

Under the experimental conditions of this study, the test item was identified as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).