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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:
2009-11-23 to 2009-12-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: OECD Guideline for Testing of Chemicals 437: Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants (September, 2009).
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: The Bovine Corneal Opacity and Permeability Assay (BCOP) was performed according to the INVITTOX (UK) protocol no. 98 "The Bovine Corneal Opacity and Permeability Assay", dated February 1994.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Mainzer Strasse 80, D65189 Wiesbaden, Germany

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[[2,2,4-trifluoro-5-(trifluoromethoxy)-1,3-dioxolan-4yl]oxy-ethanol
EC Number:
700-535-6
Cas Number:
1190931-34-0
Molecular formula:
C6F6H6O5
IUPAC Name:
2-[[2,2,4-trifluoro-5-(trifluoromethoxy)-1,3-dioxolan-4yl]oxy-ethanol
Details on test material:
- Name of test material (as cited in study report): Dioxolanic Alkohol
- Physical state: organic mono-constitient substance
- Analytical purity: > 95%
- Purity test date: no data
- Lot/batch No.: 107-31
- Expiration date of the lot/batch: 31 December 2020
- Stability under test conditions: not relevant
- Storage condition of test material: In the refrigerator at 2 to 8 °C
- Other:

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Freshly isolated bovine cornea. Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
Freshly isolated bovine eyes were collected from the abattoir. Excess tissue was removed from the excised eyes and they were contained and transported in Hank’s BSS supplemented with streptomycin / penicillin at room temperature. The corneae were isolated immediately after delivery of the eyes to the laboratory.


ENVIRONMENTAL CONDITIONS
N.A.

IN-LIFE DATES: From: To:
N.A.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
other: Three cornea used for each positive and negative controls
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1 ml per cornea
- Concentration (if solution): N.A.


VEHICLE
N.A.
Duration of treatment / exposure:
The incubation time lasted ten minutes (± 30 seconds).
Observation period (in vivo):
Opacity was measured imediately after rinsing off the test item from the treated cornea, and again 2 hours thereafter.
After completion of the opacity measurements, the cornea was incubated for another 90 min before measuring the permeability.
Number of animals or in vitro replicates:
Three test runs: Test item each was investigated by using three cornea
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): After the test item or control items, respectively, were rinsed off from the application side by changing cMEM several times, in minimum three times.
- Time after start of exposure: Fresh cMEM was added and opacity was measured (t10). The corneae were then incubated at 32 ± 2 °C for further two hours in a vertical position, followed by a third opacity reading (t130).


SCORING SYSTEM:

The following formula was used to determine the in vitro score of the negative control:
In vitro Score = opacity value + (15 x OD490 value)
The following formula was used to determine the in vitro score of the positive control and the test item:
In vitro Score = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The in vitro score was calculated for each individual treatment and positive control cornea.
The mean in vitro score value of each treated group was calculated from the individual in vitro score values.

Depending on the score obtained, the test item was classified into one of the following categories:
0 - 3: Non eye irritant
3.1 - 25: Mild eye irritant
25.1 - 55: Moderately eye irritant
55.1 - 80: Severe eye irritant
>80.1: Very severe eye irritant

TOOL USED TO ASSESS SCORE: hand-slit lamp / biomicroscope / fluorescein
The opacitometer determines changes in the light transmission passing through the corneae.
For permeability determination fluorescein solution in HBSS was used (measurement via a spectro photometer)

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
10 min exposure to test item; opacity measurement 2 hours after rinsing off the test item
Value:
-0.33
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Negative controls with a mean opacity score of 0
Positive controls validity:
valid
Remarks:
Positive controls with mean opacity score 62.33
Irritation parameter:
fluorescein leakage
Run / experiment:
10 min exposure to test item. Measurement of permeability 90 min after completion of opacity determination (which itself was conducted 2 hours after exposure)
Value:
0.067
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Negative control with a mean permeability score of 0.058
Positive controls validity:
valid
Remarks:
Positive controls with mean permeability score of 1.682
Irritation parameter:
in vitro irritation score
Run / experiment:
Combined score of both opacity and permeability determinations
Value:
0.68
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Negative controls with a mean score of 0.87
Positive controls validity:
valid
Remarks:
Positive controls with a mean score of 87.56
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no damage reported

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, no increase of opacity and no permeability of the cornea was observed
- Acceptance criteria met for positive control: yes, clear opacity and distinct permeability of the cornea
- Range of historical values if different from the ones specified in the test guideline:
Historical control data:
positive control mean value 91.72, +/- 41.27
negative control mean value: 2.03 +/- 1.06

Any other information on results incl. tables

The test item showed effects which were virtually the same compared to teh negative controls. The high positive control results give proof on the validity of the study.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item Dioxolanic alcohol did not cause any opacity or permeability of the corneae compared with the results of the negative control. The calculated mean in vitro score was 0.68 and therefore, the test item was considered as non corrosive to eyes and . In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item Dioxolanic alcohol is not requiring classification for eye irritation or serious eye damage according to the CLP.
Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of Dioxolanic alcohol by means of the BCOP assay using fresh bovine corneae. The study followed the protocol according to OECD Guideline 437.

After a first opacity measurement of the fresh bovine corneae (t0), the neat test item Dioxolanic alcohol%, the positive, and the negative controls were applied to corneae and incubated for 10 minutes at 32 ± 2 °C while the posterior chamber was filled with MEM medium supplemented with sodium bicarbonate and L-glutamine and 1% fetal calf serum (FCS) (complete medium = cMEM). After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae and opacity was measured again (t10). Further, the corneae were incubated for another 120 minutes at 32 ± 2 °C in complete medium, and opacity was measured a third time (t130). After the opacity measurements permeability of the corneae was determined while application of 1 mL of a fluorescein solution for 90 minutes at 32 ± 2 °C in a horizontal position. The coming out liquid was measured spectrophotometrically. With the negative control (0.9% NaCl solution) neither an increase of opacity nor permeability of the corneae could be observed. The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae and therefore, is classified as very severe eye irritant.

The test item Dioxolanic alcohol did not cause any opacity or permeability of the corneae compared with the results of the negative control. The calculated mean in vitro score was 0.68 and therefore, the test item was classified as non eye irritant. In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item Dioxolanic alcohol is not considered to be an eye irritant.