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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
Adequacy of study:
key study
Study period:
2016-08-05
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 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)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Certificate issued by "The Department of Health of the Government of the United Kingdom".

Test material

Reference
Name:
Unnamed
Test material form:
liquid

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: adult cattle
- Characteristics of donor animals (e.g. age, sex, weight): 12 to 60 months old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): After slaughter eyes were excised by an abattoir employee and placed in Hanks' Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100IU/mL and streptomycin at 100 µg/mL). Transported to test facility over ice packs on the day of slaughter. The corneas were prepared immediately on arrival.
- indication of any existing defects or lesions in ocular tissue samples: Before and after dissection, all eyes were macroscopically examined. Only corneas free of damage were used.
- Indication of any antibiotics used: HBSS supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL).

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): used as provided

Duration of treatment / exposure:
The undiluted item applied for 10 minutes.
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS / QUALITY CHECK OF THE CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of
damage were used. The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32+/- 1 °C for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

NUMBER OF REPLICATES
three replicates for negative controls, positive controls and test samples, each.

NEGATIVE CONTROL USED
Three negative controls were used.

POSITIVE CONTROL USED
Three positive controls were used.

APPLICATION DOSE AND EXPOSURE TIME
Test item was used as provided (undiluted). The exposure time was 10 minutes at 32+/- 1°C.

POST-INCUBATION PERIOD: 90minutes at 32 +/- 1 °C: Evaluation if the permeability of the corneas to sodium fluorescein.

REMOVAL OF TEST SUBSTANCE
- After the exposure period, the corneas were rinsed three times with fresh complete EMEM containing phenol red befor a final rinse with complete EMEM without phenol red. Then, fresh EMEM without phenol red was added to the corneas.

- POST-EXPOSURE INCUBATION:
60 minutes at 32 +/- 1 °C

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Calculated by subtracting the inital opacity reading from the final opacity reading. Values were then corrected by subtracting the average change in opacity observed for the negative control corneas. Then the mean opacity value of each treatment group was calculated.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a
microtiter plate reader (Anthos 2001) was measured at a wavelenght of 492 nm (OD492).

- Others:
Histopathology: the corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Value:
2.6
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Value:
2.3
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: permeability measurement (OD)
Value:
0.015
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test item were clear post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The positive control In Vitro Irritancy Score was within the range of 29.6 to 52.0. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for positive control: The negative control gave opacity of <2.9 and permeability <0.103. The negative control acceptance criteria were therefore satisfied.
- Range of historical values if different from the ones specified in the test guideline:

Any other information on results incl. tables

Table 1: Individual and Mean Corneal Opacity and Permeability Measurements

Treatment Cornea Number Opacity Permeability (OD) In Vitro Irritancy Score
Pre- Treatment Post- Treatment Post Incubation Post-Incubation-Pre- Treatment Corrected Value   Corrected Value
Negative Control 1 3 3 4 1   0.069    
3 3 4 5 2   0.005    
5 3 4 4 1   0.017    
  1.3a   0.030c   1.8
Positive Control 2 2 27 31 29 27.7 1.373 1.343  
4 4 40 37 33 31.7 1.204 1.174  
6 3 28 31 28 26.7 1.198 1.168  
  28.7b   1.228b 47.1
Test Item 7 3 3 8 5 3.7 0.076 0.046  
8 4 6 6 2 0.7 0.017 0.000  
9 5 5 9 4 2.7 0.018 0.000  
  2.3b   0.015b 2.6

a = mean of the post-incubation – pre-incubation values

b = mean corrected value

c = mean permeability

Applicant's summary and conclusion

Interpretation of results:
other: EU GHS (CLP) criteria not met
Remarks:
not classified
Conclusions:
The test item score of 2.6 for the in vitro irritancy score was more than the negative control and less than the positive control.
Executive summary:

The purpose of this in vitro test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage in accordance with OECD Guideline 437 and EU Method B.47 and in compliance with GLP criteria. The Bovine Corneal Opacity and Permeability (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. The undiluted test item was applied for 10 minutes followed by an incubation period of 120 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). The positive control In Vitro Irritancy Score was within the range of 29.6 to 52.0. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of <2.9 and permeability <0.103. The negative control acceptance criteria were therefore satisfied. The corneas treated with the test item were clear post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

The in vitro irritancy scores for corneas exposed to the test item was 2.6. The mean cornea opacity score for corneas exposed to the test item was 2.3. The mean permeability measurement (OD) for corneas exposed to the test item was 0.015.