Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

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)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
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Constituent
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Constituent
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Constituent
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Constituent
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Constituent
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Constituent
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Constituent
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Constituent
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Constituent
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Name: Baccartol Crude
Batch no.: VE00364716
Appearance: yellow liquid
Composition: OILS, CITRONELLA, REACTION PRODUCTS WITH ACETONE
Purity: Substance is a UVCB - cannot be determined
Expiry date 02. Apr. 2017
Storage: Room Temperature (20 ± 5°C); Keep away from light; Dry, well ventilated, preferably full, hermetically sealed.

Test animals / tissue source

Species:
other: Bos primigenius Taurus (fresh bovine corneas)
Strain:
not specified
Details on test animals or tissues and environmental conditions:
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 Hank’s balanced salt solution (supplemented with 0.01% streptomycin and 0.01% penicillin). Then, the corneas were dissected and incubated in medium at 32 ± 1 °C in an incubation chamber for 1 h.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
750 μL
Duration of treatment / exposure:
10 mins
Duration of post- treatment incubation (in vitro):
2 hours
Number of animals or in vitro replicates:
3
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 without phenol red (32 ± 1 °C) was filled. The holders were then incubated for 1 h in the incubation chamber at 32 ± 1 °C.

Experimental Parameters
Date of treatment: 14. Apr. 2016
Incubation time: 10 min
Negative control: 0.9 % sodium chloride solution
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.
The baseline opacity was measured by placing the holder with the cornea in a spectral photometer and recording the absorbance at 570 nm.
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 resp. test item resp. positive control were applied to each replicate.
According to the characteristics of the test item, the following treatment procedure was
performed:

Closed Chamber Method
The “closed chamber-method” is used for liquid substances.
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 min 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 h at 32 ± 1 °C (post-incubation).
After the post-incubation, the cMEM without phenol red was renewed in both chambers. Then, the final opacity value of each cornea was recorded (again by measurement at 570 nm). 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 min at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, the permeability of the cornea was measured as optical density of the liquid at 490 nm.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
2.71
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
In the negative control, no signs of eye irritation were observed.
The positive control induced showed effects on the cornea of the bovine eye.
The test item Baccartol Crude showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 2.71.

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:
GHS criteria not met
Conclusions:
This in vitro study was performed to assess corneal damage potential of Baccartol Crude by quantitative measurements of changes in opacity and permeability in a bovine cornea. The test item Baccartol Crude was brought onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 h and whose opacity had been determined. The test item was incubated on the cornea for 10 min at 32 ± 1 °C. After removal of the test item and 2 h post-incubation, opacity and permeability values were measured.
The test item was tested pure.
Under the conditions of this test, the test item Baccartol Crude showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 2.71.
According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.
The negative control (physiological sodium chloride solution) 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.