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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
Remarks:
.
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
No deviations from the study plan were observed.

Data source

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

Materials and methods

Test guideline
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)
Version / remarks:
OECD Guideline for the Testing of Chemicals, Method No. 437, adopted 09. Oct. 2017: “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
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP-Conformity All procedures according to the principles of GLP (Chemikaliengesetz §19a and §19b and annexes 1 and 2 from 28. Aug. 2013, published in Federal Law Gazette, Germany (BGBl) No. 55/2013 as of 06. Sep. 2013, and further revisions).

Test material

Constituent 1
Test material form:
liquid: viscous
Details on test material:
- State of aggregation: contains polymer which is not soluble after dessication
- Particle size distribution: not appropriate (liquid)
- Mass median aerodynamic diameter (MMAD): not appropriate
- Geometric standard deviation (GSD): not appropriate
- Shape of particles: not appropriate
- Surface area of particles: not appropriate
- Crystal structure: not appropriate
- Coating: not appropriate
- Surface properties: surface tension see appropriate section of IUCLID
- Density: see appropriate section of IUCLID
- Moisture content: see above
- Residual solvent: see above
- Activation: not appropriate
- Stabilisation: not required as long as treated properly and instructions of manufacturer are followed
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: The test material is representative of the registered substance
- Expiration date of the lot/batch: not relevant
- Purity test date: not relevant

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Species Bos primigenius Taurus (fresh bovine corneas)
Bovine corneas were used. They were collected from slaughtered cattle that were between 12 and 60 months old:
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container within 1 hour 15 minutes.
The following Guideline was applied: OECD Guideline for the Testing of Chemicals, Series on Testing and Assessment No. 160: “GUIDANCE DOCUMENT ON “THE BOVINE CORNEAL OPACITY AND PERMEABILITY (BCOP) AND ISOLATED CHICKEN EYE (ICE) TEST METHODS: COLLECTION OF TISSUES FOR HISTOLOGICAL EVALUATION AND COLLECTION OF DATA ON NON-SEVERE IRRITANTS”; 25. Oct. 2011
Additional information was taken from:
• “Bovine Corneal Opacity and Permeability (BCOP) Assay”, SOP of Microbiological Associates Ltd., UK, Invittox (UK) protocol no. 124, Procedure Details, April 1997, last update Aug. 1999; based on Gautheron et al. (1992), refined by Vanparys et al. (1994)
• “The Bovine Corneal Opacity and Permeability Assay – Method of Gautheron”; Invit-tox (UK) protocol no. 98, April 1996
• Commission Regulation (EU) 2017/735 amending Regulation (EC) No. 440/2008, EU Method B.47 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, adopted 14. Feb. 2017


Test system

Vehicle:
unchanged (no vehicle)
Remarks:
The test item is a liquid substance. It was tested directly, without dilution or preparation of a solution. The test item was shaken before use.
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Chemicals
• Minimum Essential Medium (MEM) without phenol red
• Minimum Essential Medium (MEM) with phenol red
• L-glutamine
• Sodium bicarbonate NaHCO3
• 1% fetal calf serum
• cMEM without phenol red:
495 mL MEM without phenol red are supplemented with 5 mL FCS, 146 mg L-Glutamine and 0.55 g NaHCO3
248 mL MEM without phenol red are supplemented with 2.5 mL FCS, 73 mg L-Glutamine and 0.28 g NaHCO3
(as soon as the MEM without phenol red is supplemented with FCS and L-Glutamine and NaHCO3, it is designated in the following as cMEM (= complete MEM) without phenol red)
• cMEM with phenol red:
495 mL MEM with phenol red are supplemented with 5 mL FCS and 146 mg L-Glutamine (as soon as the MEM with phenol red is supplemented with FCS and L-Glutamine, it is designated in the following as cMEM (= complete MEM) with phenol red)
• Sodium fluorescein (C20H11O5Na)
• Hank’s Balanced Salt Solution (HBSS) with Penicillin-Streptomycin solution (transport medium)
• Hank’s Balanced Salt Solution (HBSS) without Penicillin-Streptomycin solution (negative control)
• Penicillin-Streptomycin solution
Duration of treatment / exposure:
1 h + 10 min
Duration of post- treatment incubation (in vitro):
90 min
Number of animals or in vitro replicates:
0 animals
3 in vitro replicates
Details on study design:
Purpose and Principle of the Study
Thisin vitro study was performed to assess the corneal damage potential of test item by quantitative measurements of changes in opacity and permeability in a bovine cornea. The study was performed for regulatory purposes.
The BCOP test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine corneain vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. Both measurements are used to calculate an “In Vitro Irritancy Score (IVIS)”, which is used to classify the test item in the UN Globally Harmonised System (GHS).
The BCOP test method uses isolated corneas from the eyes of freshly slaughtered cattle. Corneal opacity is measured quantitatively as the amount of light transmission through the cornea. Permeability is measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea, as detected in the medium in the posterior chamber. Test item is applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder.

Results and discussion

In vitro

Results
Irritation parameter:
cornea opacity score
Remarks:
Mean Opacity Difference corrected
Run / experiment:
Opacity Values Test Item and Positive Control: Mean Opacity Difference corrected
Value:
ca. 85.84
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Hank’s Balanced Salt Solution (HBSS) 0.11 Mean Opacity Difference
Positive controls validity:
valid
Remarks:
Dimethylformamide (DMF) 88.45 Mean Opacity Difference corrected
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
For the permeability measurement, three replicates for each treatment group were measured three times. cMEM without phenol red was measured as blank value as well. The optical density values at 492 nm are given in the following tables

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
test item induced serious eye damage on the cornea of the bovine eye. The calculated IVIS (In Vitro Irritancy Score) is 85.84.
According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 55 induces serious eye damage, that should be classified as UN GHS Category I.
Executive summary:

In a Study following OECD Guideline 437, cornea of bovine eyes were incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and exposed to the test item for 10 minutes at 32 ± 1 °C. After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured.

Hank’s Balanced Salt Solution (HBSS) was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (In Vitro Irritancy Score) is 0.17.

Dimethylformamide (DMF) undiluted was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated IVIS is 85.84.

Under the conditions of this study, the test item induced serious eye damage on the cornea of the bovine eye. The calculated IVIS is 88.45.

According to OECD Guideline 437 (Oct. 2017), a substance with an IVIS > 55 induces serious eye damage, that should be classified as UN GHS Category I