Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 30th August 2018 to 13rd September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

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 09. Oct. 2017
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) 2017/735 amending Regulation (EC) No. 440/2008.
Adopted 14. Feb. 2017
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Details on test material:
The test material is a UVCB substance.
Specific details on test material used for the study:
Name: ACID BROWN 396:1
Batch no.: 77171
Appearance: Brown powder
Purity: 100% (UVCB)
Homogeneity: homogeneous
Storage: Room temperature (20 ± 5 °C)
EINECS-No.: 947-779-0

STORAGE
The test item was stored in the test facility in a closed vessel at room temperature (20±5°C).

PREPARATION OF TEST ITEM
The test item is a solid non-surface-active substance and has been tested neat.

Test animals / tissue source

Species:
other: Bos primigenius Taurus (fresh bovine corneas)
Details on test animals or tissues and environmental conditions:
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, 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 in a suitable cooled container within 1 hour.

Test system

Vehicle:
Hank's balanced salt solution
Amount / concentration applied:
The following amounts of the test item were tested neat and applied directly on the cornea using a weighing funnel:
Replicate Amount
1 502.1 mg
2 500.0 mg
3 506.7 mg
Duration of treatment / exposure:
Exposure time of the controls and test item on the corneas was 4 hours.
Number of animals or in vitro replicates:
3 replicates
Details on study design:
TEST VESSELS
All vessels used are made of glass or sterilised plastic. The glassware was sterilised before use by auto-claving. Schott bottles, glass vials, and culture flasks for solutions and media were used.

METHOD DESCRIPTION
After the initial incubation, the medium was completely changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used. For each treatment group (negative control solution, test item and positive control solution), three replicates were used.
According to the characteristics of the test item, the following treatment procedure was performed:
- Open chamber method, in order to apply the test item and record the final opacity value. The test item could be applied directly onto the cornea. The test item was given on the epithelium in such a manner that the cornea was evenly covered with test item.
- Permability test, after the recording of the final opacity value,for the detection of permeability of the corneas.
The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C in a horizontal position. After incubation, the content of the posterior chamber was thoroughly mixed and pipetted in a 96-well plate. Then, its optical density at 492 nm (OD492) is measured with the microtiter plate photometer.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
ca. 73.9
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
CALCULATION OF OPACITY
The change of opacity value of each treated cornea with test item, positive control and negative control was calculated by subtracting the initial basal opacity from the post treatment opacity reading for each cornea. The average change in opacity of the negative control cornea was calculated and this value was subtracted from the change in opacity of each treated cornea with test item and positive control to obtain a corrected opacity.

CALCULATION OF PERMEABILITY
The corrected OD492 value of each cornea treated with test item and positive control was calculated by subtracting the mean negative control cornea value from the original permeability value for each cornea. The mean OD492 value for each treatment group (test item, positive control and negative control) was determined by averaging the final corrected OD492 values of the treated corneas for one treatment group.

CALCULATION OF IVIS (In Vitro Irritancy Score)
The IVIS of each replicate of the negative control was calculated from the following equation:
IVIS = opacity difference + (15 x corrected OD492 value)
The IVIS of each replicate of the positive control and of the test item were calculated from the following equation:
IVIS = (opacity difference – mean opacity difference of the negative control) + [15 x (OD492 – mean OD492 of the negative control)].

Any other information on results incl. tables

IVIS VALUES

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

Test group

 Mean IVIS

 Negative control HBSS

 2.18

 Test item ACID BROWN 396:1

 73.90

Positive control 20% imidazole solution  

 125.44

VALIDITY

The test is considered as valid if the positive control causes an IVIS that falls within two standard deviations of the current historical mean. The mean IVIS of the negative control has to show an IVIS ≤ 3.

The validity criteria are the following:

- Mean IVIS of negative control HBSS ≤ 3;

- Mean IVIS of positive control 20% imidazole solution between 79.15 – 146.86.

Since the IVIS of negative control HBSS is 2.18 and the IVIS of the positive control 20% imidazole solution is 125.44, the validity criteria are fullfilled.

ASSESSMENT

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.

The classification scheme is:

IVIS

UN GHS

 ≤ 3

No category  

 > 3 and ≤ 55

No prediction can be made  

 > 55

 Eye damage Category I

By this table, the test item ACID BROWN 396:1 induced serious eye damage on the cornea of the bovine eye because is IVIS (73.90) was >55.

The experiment is considered as sufficient for the classification of the test item, because all three replicates of the test item lead to the same assessment for the test item.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Remarks:
IVIS > 55
Conclusions:
This in vitro study was performed to assess corneal damage potential of ACID BROWN 396:1 by quantitative measurements of changes in opacity and permeability in a bovine cornea.
Under the conditions of this test, the test item ACID BROWN 396:1 induced serious eye damage on the cornea of the bovine eye. The calculated mean IVIS was > 55. 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.
The test is considered valid and sufficient for the classification of the test item, because all three replicates of the test item lead to the same assessment.
Executive summary:

This in vitro study was performed to assess the corneal damage potential of ACID BROWN 396:1 by quantitative measurements of changes in opacity and permeability in a bovine cornea.

The 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. 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.

Bovine corneas were collected from slaughtered cattle that were between 12 and 60 months old.

The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, 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 mean IVIS (In Vitro Irritancy Score) was 2.18. The positive control induced serious eye damage on the cornea with a calculated mean IVIS of 125.44.

Under the conditions of this study, the test item ACID BROWN 396:1 induced serious eye damage on the cornea of the bovine eye. The calculated mean IVIS was 73.90.

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.