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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:
18 March 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
(2013)
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:
(2010)
Deviations:
no
Principles of method if other than guideline:
- The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006.
- In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.
- Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
liquid
Details on test material:
- Name of test material (as cited in the expert statement): Folenox
- Chemical name: 4,4,8,8-Tetramethyloctahydro-4a,7-methanonaphtho[1,8a-b]oxirene (IUPAC)
- Empirical formula: C15H24O
- Structural formula: See IUCLID section 1

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
Bovine eyes from young cattle were obtained from the slaughterhouse, where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Test system

Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 750 µl per cornea

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µl of physiological saline per cornea

POSITIVE CONTROL
Amount(s) applied (volume or weight with unit): 750 µl per cornea
Concentration: 10% (w/v) Benzalkonium Chloride in physiological saline


Duration of treatment / exposure:
10 minutes
Number of animals or in vitro replicates:
Three corneas / substance or control
Details on study design:
TEST SITE
- Isolated bovine cornea

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 10 minutes

SCORING SYSTEM:
- After exposure the cornea is thoroughly rinsed to remove the test substance followed (possible pH effects recorded) by incubation for 120 minutes. Opacity was measured and permeability of the cornea was evaluated.

- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).
Opacity and permeability values were also evaluated independently.

TOOL USED TO ASSESS SCORE:
- opacitymeter and microplate reader

DATA EVALUATION:
A test substance that induces an IVIS ≤ 3 is defined as non irritant, IVIS > 3 ≤ 55 mild to moderate (no prediction for classification can be made) and IVIS ≥ 55 is defined as a corrosive or severe irritant (cat.1).

Acceptability of the assay:
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within the laboratory historical mean value.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Results and discussion

Results of ex vivo / in vitro study
Irritation parameter:
in vitro irritation score
Value:
0.6
Irritant / corrosive response data:
The corneas treated with Folenox showed opacity values (corrected) ranging from 0 to 1 and permeability values ranging from -0.006 to -0.002. The corneas were clear after the 12 minutes of treatment with Folenox. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -0.1 to 1.0 after 12 minutes of treatment with Folenox.
Other effects:
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 117 and was within the historical positive control data range. Negative and positive controls were valid and It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The substance Folenox is not irritant or corrosive in the Bovine Corneal Opacity and Permeability test performed according to OECD 437 guideline and GLP principles.
Executive summary:

The substance Folenox was screened for the eye irritation potential using the Bovine Corneal Opacity and Permeability test performed according to OECD 437 guideline and GLP principles.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 117 and was within the historical positive control data range. Negative and positive controls were valid and it was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Folenox did not induce ocular irritation through both endpoints, opacity and permeability, resulting in a mean in vitroirritancy score of 0.6 after 12 minutes of treatment.Based on the results, it is concluded that Folenox is not irritant or corrosive in the Bovine Corneal Opacity and Permeability test. Since Folenox induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.