Registration Dossier

Toxicological information

Eye irritation

Currently viewing:

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:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
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:
(Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Fatty acids, C8-10, mixed esters with adipic acid and trimethylolpropane
- Physical state: yellowish brown liquid
- Analytical purity: 100%
- Lot/batch No.: OE 10428
- Expiration date of the lot/batch: 2013-04-28
- Storage condition of test material: in a closed vessel at room temperature (20 ± 5 °C)

Test animals / tissue source

Species:
other: cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST METHOD
The bovine corneal opacity and permeability (BCOP) test is an in-vitro test method used to classify substances as “ocular corrosives and severe irritants”. The potential of a test substance to cause ocular corrosivity or severe irritancy is measured by its ability to induce opacity and increased permeability in isolated bovine corneas. The opacity and permeability assessments of the corneas are combined to derive an in-vitro irritancy score (IVIS), which is used to classify the irritancy level of the test substance.

IDENTIFICATION OF THE SOURCE OF THE EYES, STORAGE AND TRANSPORT CONDITIONS
- Source: Müller Fleisch GmbH, Birkenfeld, Germany
- Donor animals: 12-60 months
- Transport medium and temperature conditions: Hank’s balanced salt solution (HBSS) supplemented with 0.01% [v/v] penicillin/streptomycin

PREPARATION OF THE EYES (BEFORE EXPOSURE)
- Eyes free of defects (scratches, neovascularisation): yes
- Dissection of the eyes and treatment: corneas were excised with a scalpel and cut from the globe with a 2 to 3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder, in which pre-warmed complete Minimum Essential Medium (cMEM) without phenol red was filled.
- Description of the cornea holder: the cornea holders consisted of an anterior and a posterior compartment, which interface with the epithelial and endothelial sides of the cornea.
- Test medium and temperature conditions used in the cornea holder: complete Minimum Essential Medium (cMEM) with and without phenol red, supplemented with foetal bovine serum, L-glutamine, sodium bicarbonate and penicillin/streptomycin; prior to use: pre-warmed to 32 ± 1 °C
- Equilibration time: 1 h at 32 ± 1 °C
- Quality check of the equilibrated corneas: free of macroscopic defects, initial opacity< 7 (range: 1.35-2.25 for all corneas)

DETERMINATION OF THE INITIAL OPACITY
- Method: corneal opacity was determined by the amount of light transmission through the cornea using a spectral photometer.
- Specification of the device: Spectral photometer Specord 205, Analytik Jena, Germany

Test system

Vehicle:
other: olive oil
Controls:
other: number of corneas for the solvent control: 3; number of corneas for the negative control: 3; number of corneas for the positive control: 3
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied in the test: 750 µL
- Concentration (if solution): 10%

SOLVENT CONTROL
- Substance: olive oil
- Amount(s) applied in the test: 750 µL

NEGATIVE CONTROL
- Substance: 0.9% sodium chloride
- Amount(s) applied in the test: 750 µL

POSITIVE SUBSTANCE
- Substance: sodium hydroxide
- Concentration (if solution): 10% in 0.9% sodium chloride
- Amount(s) applied in the test: 750 µL
Duration of treatment / exposure:
10 min at 32 ± 1 °C
Duration of post- treatment incubation (in vitro):
2 h
Number of animals or in vitro replicates:
number of corneas for the test item: 3
Details on study design:
TEST CONDITIONS
- Short description of the method used: closed-chamber method
The respective substance (negative control, positive control, solvent control or the test item solution) was applied by pipetting 750 µL of the appropriate solution through the refill hole in the holder on the cornea. The test item was applied to the epithelium of the cornea in such a manner that as much as possible of the cornea was covered with the test item solution. Each cornea was exposed for a period of 10 min at 32 °C.

POST-EXPOSURE TREATMENT
- Removal of the test substance: first, corneas were rinsed with cMEM containing phenol red followed by a final rinsing with cMEM without phenol red. Afterwards, the anterior chamber was filled with cMEM (without phenol red), and the corneas were stored for an additional 2 h at 32 °C (post-incubation).
- Medium for washing the corneas: cMEM containing phenol red
- Medium for final rinsing: cMEM without phenol red

DETERMINATION OF THE FINAL OPACITY
- Method: corneal opacity was determined by the amount of light transmission through the cornea using a spectral photometer.
- Time of determination: after the post-exposure treatment, the cMEM without phenol red was renewed in both chambers and final opacity was recorded at 570 nm.
- Specification of the device: Spectral photometer Specord 205, Analytik Jena, Germany

DETERMINATION OF THE CORNEAL PERMEABILITY:
- Method: the cMEM without phenol red was removed from the anterior chamber and fluorescein solution was added. The chambers were closed again and incubated for the respective period. After incubation, the content of the posterior chamber was thoroughly mixed, and the concentration of fluorescein solution in this chamber was measured at 490 nm using a spectral photometer.
- Amount and concentration of the dye: 1 mL sodium fluorescein solution (4 mg/mL)
- Incubation time: 90 ± 5 min at ca. 32 ± 1 °C
- Specification of the spectrophotometer: Spectral photometer Specord 205, Analytik Jena, Germany

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
10 min of exposure
Value:
0.217
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Based on the measurements for opacity and permeability, an In Vitro Irritancy Score (IVIS) ≤ 55 was calculated after treatment with the test substance. The positive and negative controls showed the expected results.

Any other information on results incl. tables

Table 1. Opacity scores after 10 min exposure and 2 h post-treatment incubation period

Treatment group

Replicate

Initial opacity

Final opacity

Opacity difference

Mean

SD

Test substance (10% in olive oil)

1

2

3

2.2496

1.6110

1.7183

2.2341

1.8754

2.2090

-0.0155

0.2644

0.4907

0.2465

0.2536

Solvent control

(olive oil)

1

2

3

1.4531

1.4904

1.4178

1.5136

1.6300

1.6527

0.0604

0.1397

0.2350

0.1450

0.0874

Negative control (0.9% NaCl)

1

2

3

1.5646

1.5449

1.5115

1.8557

1.8963

1.6734

0.2911

0.3514

0.1619

0.2681

0.0968

Positive control (10% NaOH in 0.9% NaCl)

1

2

3

1.6630

1.3465

1.5985

174.381

174.864

185.353

172.718

173.517

183.755

176.663

6.155

SD = standard deviation

Table 2. Permeability score after 10 min exposure and 2 h post-treatment incubation period

Treatment group

Replicate

OD490

Correction factor

Corrected OD490

Mean

SD

Test substance (10% in olive oil)

1

2

3

0.0048

-0.0012

-0.0009

5

5

5

0.0240

-0.0060

-0.0045

0.0045

0.0169

Solvent control

(olive oil)

1

2

3

-0.0018

-0.0003

0.0002

5

5

5

-0.0090

-0.0015

0.0010

-0.0032

0.0052

Negative control (0.9% NaCl)

1

2

3

0.0037

0.0039

0.0088

5

5

5

0.0185

0.0195

0.0440

0.0273

0.0144

Positive control (10% NaOH in 0.9% NaCl)

1

2

3

0.3579

0.3795

0.4826

5

5

5

1.7895

1.8975

2.4130

2.0333

0.3332

SD = standard deviation

Table 3. In vitro irritancy score (IVIS) after 10 min exposure and 2 h post-treatment incubation period

Treatment group

IVIS

per cornea

per group

mean

RSD [%]

Test substance (10% in olive oil)

0.569

0.644

0.822

0.217

19.2

Solvent control

(olive oil)

-0.075

0.117

0.250

0.098

166.5

Negative control (0.9% NaCl)

0.248

0.077

0.326

0.678

58.6

Positive control (10% NaOH in 0.9% NaCl)

198.883

201.302

219.272

206.486

5.4

RSD = relative standard deviation

Table 4. Validity criteria (according to OECD 437)

Parameter

Criterion

Mean value (present study)

IVIS of negative control (0.9% NaCl)

0-3

0.678

IVIS of positive control (10% NaOH)

103.3-276.1

206.486

Table 5. IVIS - Historical range of controls

Parameter

IVIS (Negative control)

IVIS (solvent control)

IVIS (Positive control)

Substance

0.9% NaCl

olive oil

10% NaOH

Mean

0.456

0.456

189.7

Standard deviation

0.773

0.352

43.2

Range (min-max)

-1.420-2.592

0.123-1.072

107.7-333.3

Range (Validity)

0-3

0-3

103.3-276.1

Applicant's summary and conclusion

Interpretation of results:
other: non-corrosive (Eye Irrit. 2 or not classified) according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the test, the test substance was shown to have no corrosive potential in the Bovine corneal opacity and permeability (BCOP) test prediction model. The result does not allow for the non-classification or classification as irritant of the test substance and therefore further evaluation and/or data generation is required.