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Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report

Materials and methods

Test guideline
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)
GLP compliance:

Test material

Chemical structure
Reference substance name:
Benzenesulfonic acid, 4-C10-13-alkyl derivs.-, salts with amines, C16-C18 (even numbered) alkyl
Molecular formula:
Benzenesulfonic acid, 4-C10-13-alkyl derivs.-, salts with amines, C16-C18 (even numbered) alkyl
Test material form:
liquid: viscous
Details on test material:
Chemical registery number : 953-378-2
Chemical name : Benzenesulfonic acid, 4-C10-13-alkyl derivs.-, salts with amines, C16-C18 (even numbered) alkyl

Based on the qualitative and quantitative information on the composition, the sample used are representative of the boundary composition shared and agreed by each registrant.

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
Placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were refrigerated on arrival and used within 24 hours of receipt.

Test system

Hank's balanced salt solution
Amount / concentration applied:
0.75 mL
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
Three corneas were also allocated to the test item.
Three corneas to the positive control item.
Three corneas to the negative control item.
Details on study design:
- Washing (if done): Was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red.

In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)

IVIS Classification
<= 3 No category. Not requiring classification
>3; <=55 No prediction of eye irritation can be made
>55 Category 1, UN GHS or EU CLP Causes serious eye damage

TOOL USED TO ASSESS SCORE: Camspec Model M108 Spectrophotometer.

Results and discussion

In vitro

Irritation parameter:
in vitro irritation score
Vehicle controls validity:
Negative controls validity:
Positive controls validity:

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
The test material was classified as non-irritant.
Executive summary:

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.
The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The IVIS of the test item is 0.9. According to the data interpretation criteria, no classification is required since the IVIS is ≤ 3.

Not requiring classification to UN GHS or EU CLP.