Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

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:
adopted July 26, 2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

1
Reference substance name:
Ichthammol
EC Number:
232-439-0
EC Name:
Ichthammol
Cas Number:
8029-68-3
Molecular formula:
unspecified
IUPAC Name:
Ichthammol (UVCB substance)
Test material form:
liquid: viscous
Details on test material:
sample from a batch from regular production released for use as an active pharmaceutical ingredient in medicinal products.
Specific details on test material used for the study:
ICHTHAMMOL Batch no. 1/17

from regular production at the registrant's manufacturing site (release date: 2017-02-20)

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
Bovine eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse (Hubert Bahlmann GmbH & Co. Versandschlachterei Spezialmischfutterwerk KG, 49699 Lindern, Germany).
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.
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 and positive control groups.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Negative control item: 0.9% sodium chloride solution
Positive control item: 1% NaOH solution in aqua ad iniectabilia
Test item (active ingredient): The test item was used undiluted

750 µL of the test or control items as recommended as suitable test volume according to OECD TG 437 were added to completely cover the cornea's epithelium in the anterior chamber.
Duration of treatment / exposure:
Exposure period: 10 minutes
Duration of post- treatment incubation (in vitro):
The corneas were incubated at 32±1°C for two hours.
Number of animals or in vitro replicates:
Three corneas were used for each treatment group (test item, negative control and positive control).
Details on study design:
The BCOP study is an in vitro test method that can be used as part of a tiered testing strategy for regulatory classification and labelling of ocular corrosives and severe irritants, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labeling of Chemicals (GHS). The ocular irritancy potential of the test item is measured by its ability to induce opacity and increase permeability in an isolated bovine cornea. From these assessments, an In Vitro Irritancy Score is derived which is used to classify the irritancy level of the test item. Thereafter, test items can be classified as inducing serious eye damage (UN GHS Category 1) and as test chemicals not requiring classification for eye irritation or serious eye damage (UN GHS no Category).

Test groups and treatement:
Three corneas were used for each treatment group (test item, negative control and positive control).

Negative control item: 0.9% sodium chloride solution
Positive control item: 1% NaOH solution in aqua ad iniectabilia
Test item (active ingredient): The test item was used undiluted
Exposure period: 10 minutes

750 µL of the test or control items as recommended as suitable test volume according to OECD TG 437 were added to completely cover the cornea's epithelium in the anterior chamber.
After the exposure period of 10 minutes the exposure solution was removed from each chamber and the epithelium was washed with EMEM containing phenol red at least three times. The open-chamber method was used. The window-locking ring and glass window from the anterior chamber were removed prior to treatment. The control or test chemical was applied directly to the epithelial surface of the cornea using a micro-pipet. 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. After rinsing, the glass window was replaced on the anterior chamber to recreate a closed system and the corneas were incubated at 32±1°C for two hours. After this post-exposure incubation period, the corneas were examined.

Examination:
Corneal injury was assessed by evaluating the opacity and permeability of the cornea. Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer resulting in opacity values measured on a continuous scale.
To determine the corneal permeability 1 mL sodium fluorescein 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. The holder was incubated in a horizontal position at 32±1°C for 90±5 minutes. The amount of sodium fluorescein that crossed from the anterior to the posterior chamber was measured quantitatively using a microplate reader (Tecan Sunrise Magellan Version 7.2 ). Measurements at 490 nm were recorded as optical density (OD490). The fluorescein permeability values were determined using OD490 values based upon a visible light spectrophotometer (Tecan Sunrise) using a standard 1 cm path length.

EVALUATION:
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.
As stated in the OECD guideline, if the test substance is not identified as an ocular corrosive or severe irritant, additional testing should be conducted for classification and labelling purposes. The BCOP test method has an overall accuracy of 79% (150/191), a false positive rate of 25% (32/126), and a false negative rate of 14% (9/65), when compared to in vivo rabbit eye test method data classified according to the UN GHS classification system. When substances within certain chemical (i.e., alcohols, ketones) or physical (i.e., solids) classes are excluded from the database, the BCOP test method has an overall accuracy of 85% (111/131), a false positive rate of 20% (16/81), and a false negative rate of 8% (4/50) for the UN GHS classification system.
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. When used for this purpose, the BCOP test method has an overall accuracy of 69% (135/169), a false positive rate of 69% (61/89), and a false negative rate of 0% (0/107), when compared to in vivo rabbit eye test method data classified according to the UN GHS classification system.

The IVIS cut-off values for identifying test chemicals as inducing serious damage (UN GHS Category 1) and test chemicals not requiring classification for irritation or serious eye damage (UN GHS No Category) are given hereafter.

IVIS UN GHS
≤ 3 No Category
> 3 and ≤ 55 No prediction can be made
> 55 Category 1

Study acceptance criteria:
A test is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean, which was updated at least every three months. The negative or solvent/vehicle control responses should result in opacity and permeability values, that are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control. In cases of borderline results in the first testing run, a second testing run should be considered (but not necessarily required), as well as a third one in case of discordant mean IVIS results between the first two testing runs. A result in the first testing run is considered borderline if the predictions from the 3 corneas were non-concordant, such that:
• 2 of the 3 corneas gave discordant predictions from the mean of all 3 corneas, OR,
• 1 of the 3 corneas gave discordant prediction from the mean of all 3 corneas AND the discordant result was > 10 IVIS units from the cut-off threshold of 55.
• If the repeat testing run corroborates the prediction of the initial testing run (based upon the mean IVIS value), then a final decision can be taken without further testing. If the repeat testing run results in a non-concordant prediction from the initial testing run (based upon the mean IVIS value), then a third and final testing run should be conducted to resolve equivocal predictions, and to classify the test chemical. It may be permissible to waive further testing for classification and labelling in the event any testing run results in a UN GHS Category 1 prediction.

The recent background data are shown below:
IVIS: lower and upper limits of acceptance according to OECD Guideline No. 437;
Opacity: upper limit of acceptance according to OECD Guideline No. 437;
Permeability: upper limit of acceptance according to OECD Guideline No. 437.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Remarks:
calculation of an in vitro irritancy score (IVIS) based on opacity and permeability values
Run / experiment:
Mean of three runs
Value:
ca. 7.5
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
Under the present test conditions ICHTHAMMOL, tested in the in vitro BCOP test method, had an IVIS value of 7.461, which is slightly above the cut-off value of 3 (UN GHS no category) and well below the cut-off value of 55 (UN GHS category 1). In view of the fact that Ichthammol is well tolerated on the skin and that no side effects are registered within the "PSUR system" (see chapter 7.4.1) this outcome is interpreted in such a way that H319, Eye Irrit. 2 is valid.

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
Under the present test conditions ICHTHAMMOL, tested in the in vitro BCOP test method, had an IVIS value of 7.461, which is slightly above the cut-off value of 3 (UN GHS no category) and well below the cut-off value of 55. In view of the fact that Ichthammol is well tolerated on the skin and that no side effects are registered within the "PSUR system" (see chapter 7.4.1) this outcome is interpreted in such a way that H319, Eye Irrit. 2 is valid