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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:
June 1st, 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 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)
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Qualifier:
according to
Guideline:
other: OECD 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)
Principles of method if other than guideline:
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”; Invittox (UK) protocol no. 98, April 1996
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Species: Bos primigenius Taurus.
- Source: fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test
- Characteristics of donor animals: the cattle were between 12 and 60 months old.
- Storage, temperature and transport conditions of ocular tissue: the eyes were transported to the test facility in Hank’s balanced salt solution with 1 % Penicillin-Streptomycin solution (Penicillin 100 U/ml, Streptomycin 100 μg/ml) in a suitable cooled container. Then, the corneas were dissected and incubated in medium at 32 ± 1 °C in an incubation chamber for 1 hour.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
2 hours
Details on study design:
SELECTION AND PREPARATION OF CORNEAS: after having carefully cleaned and sterilised the cornea holders, they were kept in the incubation
chamber at 32 ± 1 °C. On the day of the assay, the MEM (Minimum Essential Medium) without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1 % fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C. The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate. After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C. After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used.

NUMBER OF REPLICATES: three for each treatment group (negative control solution, test item and positive control).

NEGATIVE CONTROL USED: HBSS- solution: Hank’s Balanced Salt Solution (HBSS) 10-fold concentrated, diluted in demin. water (1:10), batch no.: 20170601

POSITIVE CONTROL USED: Dimethylformamide, DMF, CAS-No. 68-12-2, undiluted, batch no.: 475235719.

TREATMENT METHOD: The “closed chamber-method” is used for liquid substances. After removal of the pre-incubation medium (cMEM without phenol red), the respective substance (negative control solution, test item or positive control) was applied by pipetting 750 μl of the appropriate liquid through the refill hole in the holder on the cornea. The test item was given on the epithelium in such a manner that as much as possible of the cornea was covered with the test item. Exposure time on the corneas was 10 minutes at 32 ± 1 °C.

POST-INCUBATION PERIOD: after thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, the anterior chamber was filled with cMEM without phenol red, and the corneas were stored for additional 2 hours at 32 ± 1 °C. After the post-incubation, the cMEM without phenol red was renewed in both chambers.

POST EXPOSURE INCUBATION: the cMEM without phenol red was removed from the front chamber, and 1 ml sodium fluorescein solution (concentration: 4 mg/ml) was added to the front chamber. The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed.

METHODS FOR MEASURED ENDPOINTS: after the post-incubation, the cMEM without phenol red was renewed in both chambers. Then, the final opacity value of each cornea was recorded. The permeability of the liquid was measured with the spectrophotometer at 492 nm after the 90-minute post exposure incubation.
- 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.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) - 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)].

DECISION CRITERIA: as indicated in the OECD437.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
84.29
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: RSD = 12.68 %
Other effects / acceptance of results:
- In the negative control, no signs of eye irritation were observed.
- The positive control induced serious eye damage.
- The test item induced serious eye damage on the cornea of the bovine eye.
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.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: within the range of historical data of the test facility.
- Acceptance criteria met for positive control: within the range of historical data of the test facility.

Applicant's summary and conclusion

Interpretation of results:
other: classified in Category 1 for Eye Damage, according to the CLP Regulation (EC) No. 1272/2008
Conclusions:
The substance induces serious eye damage on the cornea of the bovine eye.
Executive summary:

The corneal damage potential of the substance was assessed by quantitative measurements of changes in opacity and permeability in a bovine cornea, according to the OECD Guideline 437. The test item was brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured. The test item was incubated on the cornea for 10 minutes at 32 ± 1 °C. After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured.

Under the conditions of this study, the test item induced serious eye damage on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) was 84.29 which is within the range for classification (according to the classification criteria of OECD 437) of the substance as causing serious eye damage.