Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 July 2016 - 27 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Eye irritation is primarily defined by the extent of corneal injury; substances which damage the superficial epithelium may cause slight irritation, whereas further penetration to the corneal stroma or endothelium may induce mild or severe irritation, respectively. The Bovine Corneal Opacity and Permeability Test (BCOP) utilises opacitometric and spectroscopic methods to quantitatively assess changes to bovine corneas, to determine ocular corrosivity and severe irritancy in vitro. Corneal opacity acts as an indicator of protein denaturation, swelling, vacuolation and tissue damage, whereas corneal fluorescein retention/leakage provides a measure of permeability.

The BCOP (OECD 437) has been validated extensively and is an accepted in vitro test method to detect eye corrosion/irritation (Category 1) and/or the absence of effects (not classified under CLP). Data from the BCOP can be used for Annex VII and Annex VIII requirements for serious eye damage/eye irritation.

Solid substances were correctly predicted as UN GHS Category 1 in international validation of the Bovine Corneal Opacity and Permeability Test (BCOP). Consequently, solids and partially soluble solids, such as Bismuth silicate, are not considered to be outside the applicability domain of the test method.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
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)
Version / remarks:
Adopted July 26, 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:
Commission Regulation (EU) No. 1152/2010 method B.47, published in the Official Journal of the European Union L324, dated December 09, 2010.
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: sponsor lot# 231/08/15
- Expiration date of the lot/batch: March 2020
- Purity test date: 21 January 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Composition: Bismuth oxide (Bi2O3) 83.35%; Silicon oxide (SiO) 16.63%.
- Physical characteristics: Powder
- Storage condition of test material: At ambient temperature (10 - 25°), container kept tightly closed and stored in a dry and well-ventilated place.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Vehicle: 0.9% sodium chloride solution
- Final dilution of a dissolved solid, stock liquid or gel: 20% Bismuth silicate (w/w)

FORM AS APPLIED IN THE TEST (if different from that of starting material): Suspended fine powder

OTHER SPECIFICS: The solid test item was suspended in a 0.9% sodium chloride solution with a final concentration of 20% Bismuth silicate (w/w) as recommended in the test guideline 437 for non-surfactant solids. 0.9% NaCl solution was used as the solvent control and 20% Imidazole in 0.9% NaCl solution as the positive control item.

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST MODEL
- Test model: Bovine eyes from cattle obtained from slaughterhouse at age 6-12 months
- Source: Hubert Bahlmann GmbH & Co. Versandschlachterei Speziamichfutterwerk KG 49699, Lindern, Germany
- Storage: Bovine tissues were stored in Hanks’ Balanced Salt Solution (HBSS) (Sigma Aldrich batch no# RNBD4865) containing penicillin at 100 IU/mL (GIBCO batch no# 1759447) and streptomycin at 100 µg/mL (GIBCO batch no# 1770450).
- Quality criteria: Eyes were examined for opacity, scratches and neovascularisation, only corneas from eyes free of defects were used. Prior to the assay, corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders, were also discarded.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 32 ± 1°C

Test system

Vehicle:
physiological saline
Remarks:
0.9% NaCl
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL of the 20% test item suspension in 0.9% sodium chloride solution (w/v)
- Concentration (if solution): 20%

NEGATIVE CONTROL
- Negative control: Physiological saline solution (0.9% sodium chloride solution)
- Source and lot/batch number: B. Braun Melsungen AG batch no# 161418002
- Amount(s) applied (volume or weight): 750 µL

POSITIVE CONTROL
- Positive control: Sodium dodecyl sulphate
- Source and lot/batch number: MatTek Corporation batch no# 012616TMB
- Amount(s) applied (volume or weight): 750 µL of the 20% Imidazole suspension in 0.9% sodium chloride solution (w/v)
- Concentration (if solution): 20%
Duration of treatment / exposure:
240 minutes
Duration of post- treatment incubation (in vitro):
90 ± 5 minutes
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Bovine eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse. To minimize deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 μg/mL. Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation. Only corneas from eyes free of defects were used. The quality of each cornea was also evaluated at later steps in the assay. Corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one hour equilibration period had to be discarded. The corneas were dissected with a 2 to 3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers. Beginning with the posterior chambers, the chambers were filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM), while preventing bubble formation. The corneal holder was equilibrated at 32±1°C for at least one hour.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Corneas exhibiting macroscopic tissue damage (e.g. scratches, pigmentation, neovascularisation) or an opacity >7 opacity units were discarded. The mean opacity of all equilibrated corneas was calculated by use of an opacitometer. A minimum of three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment, solvent and positive control groups.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED
- Negative control: Physiological saline solution (0.9% sodium chloride solution)
- Source and lot/batch number: B. Braun Melsungen AG batch no# 161418002
- Amount(s) applied (volume or weight): 750 µL

POSITIVE CONTROL USED
- Positive control: Sodium dodecyl sulphate
- Source and lot/batch number: MatTek Corporation batch no# 012616TMB
- Amount(s) applied (volume or weight): 750 µL of the 20% Imidazole suspension in 0.9% sodium chloride solution (w/v)
- Concentration (if solution): 20%

APPLICATION DOSE AND EXPOSURE TIME
- Amount(s) applied (volume or weight with unit): 750 µL of the 20% test item suspension in 0.9% sodium chloride solution (w/v)
- Exposure time: 240 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the exposure period of 240 minutes (the recommended exposure time for non-surfactant solids) the test item, the negative and positive controls, were removed from each chamber. Subsequently, the epithelium was washed with EMEM7 containing phenol red at least three times. Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible. The corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber. The chamber was then filled with EMEM without phenol red.

- POST-EXPOSURE INCUBATION:
- Post-exposure preparation: To determine the corneal permeability 1 mL sodium fluorescein9 solution (5 mg/mL in 0.9% sodium chloride solution) was added to the anterior chamber (epithelial surface) while the posterior chamber (endothelial surface) was refilled with fresh EMEM.
- Incubation period: 90 ± 5 minutes
- Examination of corneas: Sodium fluorescein measurements (Tecan Sunrise Magellan Version 6.4)

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer8 resulting in opacity values measured on a continuous scale.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of (Tecan Sunrise Magellan Version 6.4 Microtiter plate reader) (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
After correcting the opacity and mean permeability (OD490) values for background opacity and the negative control permeability OD490 values, the mean opacity, and permeability OD490 values for each treatment group were combined in an empirically-derived formula to calculate an in vitro irritancy score (IVIS) for each treatment group as follows:
IVIS = mean opacity value + (15 x mean permeability OD490 value)
The opacity and permeability values were also evaluated independently to determine whether the test item induced corrosivity or severe irritation through only one of the two endpoints.

DECISION CRITERIA:
A test item that induces an IVIS > 55 is defined as a corrosive or severe irritant. The BCOP test method can also be used to identify chemicals that do not require classification for eye irritation or serious eye damage under the UN GHS classification system. The IVIS cut-off values for identifying test chemicals not requiring classification for irritation or serious eye damage (UN GHS No Category) is ≤3.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
mean opacity (n=3), relative to negative control
Value:
10.77
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
not determinable because of methodological limitations
Irritation parameter:
fluorescein retention score
Remarks:
(permeability)
Run / experiment:
Mean permeability score (n=3) relative to negative controls
Value:
0.001
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Calculated IVIS of 3 corneas exposed to Bismuth silicate
Value:
10.79
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
not determinable because of methodological limitations
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
- Range of historical values if different from the ones specified in the test guideline: The most recent control data for GLP BCOP studies at LPT (2014-2016) are as follows:

Negative control (0.9% NaCl): presented as mean ± standard deviation (limits of acceptance)
IVIS: 0.812 ± 0.762 (-0.712 - 2.336)
Opacity: 0.513 ± 0.788 (-1.064 - 2.089)
Permeability: 0.020 ± 0.012 (-0.005 - 0.045)

Positive control (Imidazol 20%): presented as mean ± standard deviation (limits of acceptance)
IVIS: 86.039 ± 10.607 (64.826 - 107.253)
Opacity: 63.051 ± 10.709 (41.634 - 84.468)
Permeability: 1.533 ± 0.463 (0.607 - 2.458)

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The corneas treated with the negative control item 0.9% sodium chloride solution revealed a mean opacity value of -0.385 ± 0.852 and a mean permeability value of 0.030 ± 0.006. The calculated IVIS value of 0.070 ± 0.899 was well below the cut-off value of 3 (UN GHS no category).
- Acceptance criteria met for positive control: The corneas treated with the positive control item 20% Imidazole in 0.9% NaCl solution revealed a mean opacity value of 64.395 ± 6.850 and a mean permeability value of 1.483 ± 0.223 compared to the solvent control. The calculated IVIS value of 86.645 ± 8.731 was within two standard deviations of the current historical mean and well above the cut-off value of 55.
- The acceptance criteria for the test were fulfilled.

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Remarks:
Indirect classification based upon an IVIS that was above the cut-off value of 3 (UN GHS no category) and below the cut-off value of 55 for identifying test substances as inducing serious eye damage (UN GHS Category 1).
Conclusions:
The BCOP (OECD 437) has been validated extensively and is an accepted in vitro test method to detect eye corrosion/irritation (Category 1) and/or the absence of effects (not classified under CLP). Solids and partially soluble solids, such as Bismuth silicate, are not considered to be outside the applicability domain of the test method. Consequently, data from the BCOP can be used to fulfil Annex VII and Annex VIII requirements for serious eye damage/eye irritation potential.

Under the test conditions, the calculated in vitro irritation score (IVIS) for Bismuth silicate of 10.790 ± 3.078 was above the cut-off value for no category, but below the cut-off value of 55 for test substances classified as inducing serious eye damage (Category 1). The BCOP test is not recommended for distinction between irritancy (Category 2 or 2A) and mild irritancy (Category 2B). Conducted according to the aforementioned guidelines and GLP, the BCOP passed all validity criteria and was considered to be reliable without restriction (Klimisch 1). In the absence of further in vitro and/or in vivo testing, the BCOP result suggests classification as Category 2.
Executive summary:

Eye irritation is primarily defined by the extent of corneal injury; substances which damage the superficial epithelium may cause slight irritation, whereas further penetration to the corneal stroma or endothelium may induce mild or severe irritation, respectively. Eye irritation of the test item was evaluated in the Bovine Corneal Opacity and Permeability Test (BCOP, OECD 437), which utilises measurements of corneal opacity as an indicator of protein denaturation, swelling, vacuolation and tissue damage, and corneal fluorescein retention/leakage provides a measure of permeability in vitro.

A 20% suspension of the test item was applied to bovine corneas in 750 µL of physiological saline solution (0.9% NaCl) for 4 hours, alongside positive and negative controls. The mean fluorescein retention/leakage score of the test item (mean ± standard deviation, 0.001 ± 0.014), corresponding to permeability, did not significantly vary from controls (0.020 ± 0.012, with lower and higher limits of acceptance of -0.005 and 0.045, respectively). However, the mean opacity score of 3 corneas was 10.770 ± 3.287. The calculated In Vitro Irritation Score (IVIS) of the test substance is above the cut-off value of 3 (UN GHS no category) and below the cut-off value of 55 for identifying test substances as inducing serious eye damage (UN GHS Category 1). Consequently, no direct conclusion regarding the irritant or corrosive classification for the test item can be made, and a Category 2 classification can be assumed.

According to Regulation (EC) No 1272/2008 (CLP Regulation or CLP) all available information relevant for the evaluation of the specific hazard should be considered in a weight of evidence (WoE) assessment, when the criteria cannot be applied directly (Article 9 (3), CLP). The BCOP (EU B.47; OECD 437) is an intentionally accepted in vitro method to detect serious eye damage (Category 1 under CLP) and/or absence of effects requiring classification for serious eye damage/eye irritation (i.e. not classified under CLP), as described in the Annex to the EU Test Methods (TM) Regulation (Council Regulation (EC) No 440/2008). Conducted according to the aforementioned guidelines and GLP, the BCOP passed all validity criteria and was considered to be reliable without restriction (Klimisch 1).

As specified in Section 8.2 of Column 2 of Annex VIII to the REACH Regulation, for substances manufactured or imported in quantities of ≥10 tpa in vivo testing must only be considered only if the in vitro study under Section 8.2.1 in Annex VII is not applicable for the substance, or the results of the study are not adequate for classification and risk assessment. There are currently no validated in vitro eye irritation test methods available that could be used for direct identification of Eye irritants Category 2 under CLP, thus, in order to distinguish between irritancy (UN GHS Category 2 or 2A) and mild irritancy (UN GHS Category 2B), in vivo testing would be required (OECD 405). In order to avoid in vivo testing and in line with the principles of the Replacement, Refinement and Reduction (3Rs) of the use of animals for scientific purposes, no further testing has been initiated and a Category 2 classification has been assumed.