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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:
26-September-2017 to ****
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']dioxodi-.mu.-thioxodi-, (Mo-Mo)
EC Number:
615-708-0
Cas Number:
72030-25-2
Molecular formula:
N/A
IUPAC Name:
Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']dioxodi-.mu.-thioxodi-, (Mo-Mo)
Constituent 2
Reference substance name:
Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']-.mu.-oxodioxo-.mu.-thioxodi-
Cas Number:
153128-45-1
Molecular formula:
N/A
IUPAC Name:
Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']-.mu.-oxodioxo-.mu.-thioxodi-
Constituent 3
Reference substance name:
Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']oxodi-.mu.-thioxothioxodi-
Molecular formula:
N/A
IUPAC Name:
Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']oxodi-.mu.-thioxothioxodi-
Constituent 4
Reference substance name:
Unspecified 2-ethylhexyl thiophosphate esters
Molecular formula:
N/A
IUPAC Name:
Unspecified 2-ethylhexyl thiophosphate esters
Constituent 5
Reference substance name:
Unspecified 2-ethylhexyl phosphate esters
Molecular formula:
N/A
IUPAC Name:
Unspecified 2-ethylhexyl phosphate esters
Constituent 6
Reference substance name:
Unspecified 2-ethylhexyl dithiophosphate esters
Molecular formula:
N/A
IUPAC Name:
Unspecified 2-ethylhexyl dithiophosphate esters
Test material form:
liquid
Specific details on test material used for the study:
CAS Registry Number: 72030-25-2
Purity: This substance has an Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB)
Physical state/Appearance: Dark green liquid

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised after slaughter and placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL), and 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

Vehicle:
unchanged (no vehicle)
Controls:
other: Negative control: Sodium chloride 0.9% w/v; Positive control: Ethanol
Amount / concentration applied:
0.75 mL of the test item was applied to the corneas.
Duration of treatment / exposure:
Incubated (at 32 ± 1 ºC) for 10 minutes.
Duration of post- treatment incubation (in vitro):
90 minutes.
Number of animals or in vitro replicates:
Three.
Details on study design:
Preparation of Corneas
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling, and the iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red, plugged and incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects, only corneas free of damage were used.

Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete EMEM and a pre treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.

Treatment of Corneas
The EMEM was removed from the anterior chamber of the BCOP holder and the test item or control items were applied to the appropriate corneas and each holder was incubated at 32 ± 1 ºC for 10 minutes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The chamber was then refilled with fresh complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed.
The holders were incubated at 32 ± 1 ºC for 120 minutes. After incubation, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

Application of Sodium Fluorescein
Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium was removed and replaced with sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 µL of media representing each cornea was dispensed into the appropriate wells of a pre labeled 96 well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
28
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Corneal Epithelium Condition
The corneas treated with the test item were partly cloudy post treatment and cloudy post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

Criteria for an Acceptable Test
The positive control In Vitro Irritancy Score was within the historical range of 29.6 to 65.1; the positive control acceptance criterion was satisfied. The negative control gave opacity of ≤2.3 and permeability ≤0.041; the negative control acceptance criteria were satisfied.

Test Item
The in vitro irritancy score of the test item was 28.0, based on this result, no prediction of eye irritation can be made for the test item.

Any other information on results incl. tables

In Vitro Irritancy Score

Treatment

In Vitro Irritancy Score

Test Item

28.0

0.9% (w/v) Sodium Chloride (Negative Control)

0.1

Neat Ethanol (Positive Control)

42.5

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of the BCOP test, no prediction of eye irritation can be made for the test item.
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 (0.9% (w/v) Sodium Chloride) and positive (Neat Ethanol) 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). 

 

Based on the results of the BCOP test, no prediction of eye irritation can be made for the test item.

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