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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:
15. Mar. 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
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:
OECD Guideline for the Testing of Chemicals, Method No. 437, adopted 09. Oct. 2017: “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
Guideline:
other: OECD Guideline 160
Version / remarks:
OECD Guideline for the Testing of Chemicals, Series on Testing and Assessment No. 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”; 25. Oct. 2011
Deviations:
no
Qualifier:
according to guideline
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, EU Method B.47 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, adopted 14. Feb. 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Petroleum resins
EC Number:
265-116-8
EC Name:
Petroleum resins
Cas Number:
64742-16-1
Molecular formula:
UVCB substance containing saturated aliphatic hydrocarbon and aromatic hydrocarbons
IUPAC Name:
naphthalene
Test material form:
liquid
Details on test material:
Reference Name: Petroleum Resins (Kendex 0897)
Sponsor Information: CAS Number 64742-16-1
Label Identification: LANXESS Lot # E267112117, 18 Jan 18, Kendex 0897
Quantity & Date Received: 24 Jan 18; 76.1 g (GW)
Physical Description: Brown liquid
Storage: Room temperature
Purity: See Certificate of Analysis
Stability: Not provided to testing facility
Specific details on test material used for the study:
No further details specified in the study report.

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Specification
Species Bos primigenius Taurus (fresh bovine corneas)

Origin
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, 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 (Penicillin 100 U/mL, Streptomycin 100 μg/mL) in a suitable cooled container within 1 hour 5 minutes.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
750 μL test item
Duration of treatment / exposure:
Exposure time of the test item on the corneas was 10 minutes.
Number of animals or in vitro replicates:
2 hours
Details on study design:
Preparations
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 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.

Experimental Parameters
Date of treatment: 15. Mar. 2018
Incubation time: 10 min.
Negative control: HBSS
Positive control: Dimethylformamide (undiluted)

Method Description
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. For each treatment group (negative control solution, test item and positive control), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 μL negative control solution, 750 μL test item and 750 mL positive control were applied to each replicate.
According to the characteristics of the test item, the following treatment procedure was performed:

Open Chamber Method
The “open chamber-method” is used for viscous substances.
In order to apply the test item, the window-locking ring and glass window of the anterior chamber was unscrewed to remove the glass disc.
750 μL of the test item and controls were given directly on the epithelium that the cornea was completely covered.
Exposure time of the test item on the corneas was 10 minutes at 32 ± 1 °C. 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 (post-incubation).
After post-incubation time, the cMEM without phenol red was renewed in both chambers.
Then, the final opacity value of each cornea was recorded.

Permeability Test
After the recording of the final opacity value, the cMEM without phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution was added to the front chamber for the detection of permeability of the corneas.
For the open chamber method, a sodium fluorescein solution with a concentration of 4 mg/mL was used.
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. Then, its optical density at 492 nm was measured with the microtiter plate photometer.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Test item
Value:
0.57
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
This in vitro study was performed to assess corneal damage potential of Petroleum Resins (Kendex 0897) by quantitative measurements of changes in opacity and permeability in a bovine cornea.
The test item Petroleum Resins (Kendex 0897) was applied directly on top of the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been determined. The test item was incubated on the cornea for 10 minutes at 32 ± 1 °C. After removal of the test item and 2 hours postincubation, opacity and permeability values were measured.
The test item was tested pure.
Under the conditions of this test, the test item Petroleum Resins (Kendex 0897) showed no effects on the cornea of the bovine eye. The calculated IVIS is 0.57.
Since Petroleum Resins (Kendex 0897) induced an IVIS < 3, according to OECD Guideline no. 437 (Oct. 2017), no classification is required for eye irritation or serious eye damage.
The negative control (HBSS) and the positive control (undiluted dimethylformamide) have met the validity criteria.
No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid.
Executive summary:

Title of Study: Evaluation of Petroleum Resins (Kendex 0897) in the Bovine Corneal Opacity and Permeability (BCOP) Test Method following OECD Guideline 437 and EU Method B.47

 

Findings and Results:

This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of Petroleum Resins (Kendex 0897) was tested through topical application for 10 minutes. One valid experiment was performed.

The study procedures described in this report were based on the most recent OECD guideline.

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

The test item Petroleum Resins (Kendex 0897) was applied as it is directly on top of the corneas. Each bovine eye which had been previously 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 32 ± 1 °C. After removal of the test item and 2 hours post-incubation, 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 IVIS (In Vitro Irritancy Score) is 1.50.

Dimethylformamide (DMF) undiluted was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated IVIS is 80.32.

 

Under the conditions of this study, the test item Petroleum Resins (Kendex 0897) showed no effects on the cornea of the bovine eye after 10 minutes of treatment. The calculated IVIS is 0.57.

Since Petroleum Resins (Kendex 0897) induced an IVIS < 3, according to OECD Guideline no. 437 (Oct. 2017), no classification is required for eye irritation or serious eye damage.