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Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
yes
Remarks:
Deviations not considered to affect the purpose or the integrity of the study (see below for further explanation)
Principles of method if other than guideline:
Deviation No. 1
Opacitometer Calibration: The reading recorded from calibrator number 2 was out of range. However, the values obtained for the positive control and the test item were closest to calibrators 1 (75) and 3 (225) respectively and therefore the results of this study are considered to be unaffected by calibrator number 2.

Deviation No. 2
The IVIS obtained for the positive control also fell below the mean for 2014 as given in the Study Plan. However, it is considered that the significant in vitro irritancy score obtained for the test item (356.3) and the condition of the test item treated corneas (opaque/white) is indicative of severe ocular irritation and therefore the lower then expect positive control score is considered
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Slags, sponge iron production by coal reduction in retort
EC Number:
940-884-3
Molecular formula:
Not applicable for inorganic UVCB
IUPAC Name:
Slags, sponge iron production by coal reduction in retort
Test material form:
solid

Test animals / tissue source

Species:
other: Cattle
Details on test animals or tissues and environmental conditions:
TEST SYSTEM
- Eyes from adult cattle (typically 12-60 months old) were obtained from a local abattoir
- The eyes were excised by an abattoir employee after slaughter and placed in Hanks Balanced Salt Solution supplement with antibiotics (pencillin at 100IU/mL and streptomycin at 100µg/mL)
- Eye were transported over ice pack on the same day of slaughter to the test facility
- Corneas were prepared immediately on arrival

Test system

Vehicle:
physiological saline
Controls:
other: Negative Control: 0.9% w/v sodium chloride solution. Positive Control: Imadazole used as a 20% w/v solution in 0.9% w/v sodium chloride solution
Amount / concentration applied:
TEST ITEM FORMLATION & EXPERIMENTAL PREPARATION

- The test item was prepared as a 20% w/v solution in 0.9% w/v sodium chloride solution
- The test item was formulated within 2 hours of being applied to the test system and assumed stable for this duration
- No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
- Positive control : Imadazole at 20%w/v solution in 0.9% w/v sodium chloride solution
- Negative control : 20% solution in 0.9%w/v sodium chloride solution

TEST ITEM
- 0.75mL of test item was applied on each cornea
- Concentration : 20% w/v solution in 0.9% w/v sodium chloride solution
- Positive Control 20%w/v solution in 0.9% w/v sodium chloride solution
- Negative Control : Used as supplied
Duration of treatment / exposure:
-Test item was applied to the corneas, the holder were gently tilted back and forth to ensure uniform application.
- Each holder was incubated at 32±1°C for 240 minutes.
Observation period (in vivo):
Not applicable
Number of animals or in vitro replicates:
Total: 9 corneas - 3 corneas/group for test item, negative and positive controls
Details on study design:
PREPARATION OF CORNEAS
- Only corneas free of damage were used
- The cornea from each eye was removed and immersed (epithelial side uppermost) in a dish containing HBSS.
- Corneas were mounted into cornea holders
- The anterior and posterior chambers of each holder were filled with Eagle's minimum essential mdium (MEM) without phenol red and plugged.
- The holders were incubated at 32±1°C for 1 hour.
- At the end of the incubation period each cornea was examined for defects and only corneas free of damage were used.

SELECTION OF CORNEAS
- Pre-treatment opacity reading was taken using a calibrated opacitometer
- Average opacilty for all corneas was calculated.

TREATMENT OF CORNEAS
- MEM was removed from the anterior chamber of the holder
- 0.75mL of the test item or control items were applied to the corneas and gently tilted back and forth before being incubated at 32±1°C for 240 minutes.
- At the end of the exposure period the test item and control items were removed from the anterior chamber and rinsed 3 times with MEM containing phenol red and once with MEM withough phenol red.
- an opacity reading and a visual assessment of the cornea was taken.

APPLICATION OF SODIUM FLUORESCEIN
- Permeability of the corneas to sodium flurescein was evaulated.
- Sodium fluorescein solution (5mg/mL) replaced the medium from the anterior chamber, the dosing holes were plugged and the holder incubated at 32±1°C for 90 minutes.

PERMEABILITY DETERMINATIONS
- Following incubation the medium was decanted and retained.
- 360µL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 792nm (OD492) was measured using the Anthos 2001 microplate reader.

HISTOPATHOLOGY
- Corneas were retained for possible conduct of histopathology.


Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
356.3
Negative controls validity:
valid
Positive controls validity:
other:
Remarks:
The positive control in-vitro irritation score narrowly missed the lower acceptable value of 66.9 but is considered insignificant.

Any other information on results incl. tables

Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in Table 1.

Following treatment with the test substance, the corneas were opaque/white post treatment. The corneas treated with the negative control were clear post treatment and those treated with the positive control were cloudy post treatment (see attached Table 2)

Criteria for an Acceptable Test

The positive control In Vitro Irritancy Score narrowly missed the lower acceptable value of 66.9. Therefore the positive control acceptance criterion was marginally outside the positive control range. However, it is considered that the significant in vitro irritancy score obtained for the test item (356.3) and the condition of the test item treated corneas (opaque/white) is indicative of severe ocular irritation and therefore the lower then expect positive control score is considered insignificant.

The negative control gave opacity of ≤4.1 and permeability ≤0.105. The negative control acceptance criteria were therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
The eye irriation potential of the test item was assessed in accordance with OECD Guideline 437. The in-vitro irritancy score of the test item was 356.3, therefore the test substance is considered to be classified as causing irreversible effects on the eye according to the GHS criteria.
Executive summary:

An in vitro eye irritation study was performed according to the OECD Guideline 437 and in compliance with GLP to assess the corneal damage potential of the test substance by means of the BCOP assay using fresh bovine corneae. 

The test item was applied at a concentration of 20% w/v in 0.9% w/v sodium chloride solution for 240 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).

 

Following treatment with test substance, the corneas were noted as opaque/white. The corneas treated with the positive control, 20% w/v Imidazole, were opaque and the corneas treated with the negative control, 0.9w/v% saline, were clear post treatment

 

The negative and positive controls met the acceptance criteria for this assay.

The In Vitro irritancy scores are summarized as follows:

Treatment

In Vitro Irritancy Score

Test Item

356.3

Negative Control

2.1

Positive Control

54.3

 

The test substance is predicted to have a classification of Category 1 (H318: Causes serious eye damage) according to the Regulation (EC) No 1272/2008(CLP).

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