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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:
from 27 April to 28 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
7th of September 2009.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: U.S. EPA (1996). Label Review Manual: 2nd Edition. EPA737-B-96-001. Washington, DC: U.S. Environmental Protection Agency.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: UN (2007). Globally Harmonized System of Classification and Labelling of Chemicals (GHS). New York & Geneva: United Nations Publications
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Propionaldehyde, reaction products with formaldehyde
EC Number:
701-281-9
Molecular formula:
C5H10O3
IUPAC Name:
Propionaldehyde, reaction products with formaldehyde
Details on test material:
- Name of test material: Propionaldehyde, reaction products with formaldehyde (EC 701-281-9) was former registered with identifier 3-Hydroxy-2-(hydroxymethyl)-2-methylpropionaldehyde (CAS 18516-18-2)
- Lot/batch No.: CH 138067/001 as cited in report freeze-dried for analytical purpose
- Expiration date of the lot/batch: 30 November 2010
- Stability under test conditions: min. 1 year
- Storage condition of test material: Room temperature

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Slaughterhouse Fröch, Kirchenplatz 3, A-7023 Zemendorf.
- Age at study initiation: 18 – 20 month and free of macroscopically visible defects.
Corneas: Isolated corneas from the eyes of cows and bulls aged between 18 – 20 month and free of macroscopically visible defects.
Justification: According to the guidelines.
Number of corneas: Total of 9 corneas: 3 for the test substance, 3 for the negative control and 3 for the positive control.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL per cornea
- Concentration (if solution): The test substance was administered undiluted as received. No analyses of the test substance were performed.
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
2 hours
Number of animals or in vitro replicates:
3
Details on study design:
NUMBER OF REPLICATES
3

DETAILS
Bovine corneas were isolated and mounted in cornea holders and equilibrated for one hour to achieve normal metabolic activity. After exclusion of corneas which did not achieve quality criteria, the corneas were distributed into groups (3 per group) and exposed to the test substance, the negative control and the positive control for 10 minutes. Then the substances were removed and the corneas were accurately washed. After a post exposure incubation of two hours the opacity and permeability of each cornea were recorded. Opacity was measured quantitatively with the aid of an opacitometer. Permeability was determined by the amount of sodium fluorescein dye that penetrated all cornea layers. For this purpose fluorescein solution was filled into the anterior chambers of the cornea holders followed by an incubation period of 90 minutes. The amount of sodium fluorescein that crossed into the posterior chambers was quantitatively measured with a spectrophotometer at OD490. Using opacity and permeability data an IVIS was calculated. The positive and negative control groups were simultaneously used for other, concurrently performed studies.

NEGATIVE CONTROL USED
No treatment.

POSITIVE CONTROL USED
1 % sodium hydroxide in deionised water (sterile).

APPLICATION DOSE AND EXPOSURE TIME
750 µl test substance were introduced into the anterior chamber through the dosing holes on the top surface of the chamber, and the holes were subsequently sealed during exposure (close-chamber method). Then the corneas were exposed in a horizontal position for 10 minutes while ensuring that the test substance adequately covered the epithelial surface. Incubation temperature, monitored with a min / max thermometer, was 29.3 °C – 33.7 °C.

POST-INCUBATION PERIOD: yes
After the exposure period substances were removed from the anterior chamber and the epithelium was washed three times with EMEM+ to determine the effectiveness of rinsing acidic or alkaline materials. cEMEM was used as a final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. Both chambers were then refilled with fresh cEMEM. The corneas were incubated for additional two hours. Incubation temperature, monitored with a min / max thermometer, was 29.3 °C – 33.7 °C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The substances were removed and the corneas were accurately washed.
- POST-EXPOSURE INCUBATION: After a post exposure incubation of two hours the opacity and permeability of each cornea were recorded.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity was determined by the amount of light transmission through the cornea. Corneal opacity was measured quantitatively in Lux with the aid of an opacitometer-kit BASF-OP2.0 (see Appendix 2), which was calibrated with a standard filter set before the corneas were measured.
Opacity values were calculated as follows:
Opacity value = ax (I0/I) + b
I = illuminance (Lux) with the cornea
I0 = illuminance (Lux) without cornea
a, b = equipment-specific variables
The standard deviation for each group was calculated as well.

- Corneal permeability:
1 mL sodium fluorescein solution was added to the anterior chamber of the cornea holder and then incubated in a horizontal position for 90 minutes. Incubation temperature, monitored with a min / max thermometer, was 29.3 °C – 33.7 °C. Passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
Permeability was determined by the amount of sodium fluorescein dye that penetrated all corneal cell layers. The amount of sodium fluorescein that crossed into the posterior chamber was quantitatively measured with the aid of a Bio-Tek EL800 microtiter plate reader at 490nm. For measurement 360 µl of cEMEM from the posterior chamber were transferred into the wells of a 96-well microtiter plate (triplicates). Data were recorded as OD490 values which were equivalent to the OD490 values based upon a visible light spectrophotometer using a standard 1 cm path length.
To proof that the measurement was performed in the linear range wells containing ten concentrations (ranging from 0.625 µg/ml to 40 µg/ml) of fluorescein solutions were additionally prepared.
To ensure that the permeability values measured were in the exponential range the linearity was determined by triple measurement of fluorescein solutions of ten concentrations. A linear calibration function and the correlation coefficient thereof was calculated.
As the OD490 values of corneas treated with 1 % sodium hydroxide lied outside of the linear range dilutions of 1:5 were measured.
The standard deviation for each group was calculated as well.


SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The mean opacity and mean permeability values were corrected for background opacity and the negative control permeability values. The mean opacity and permeability values for each treatment group were combined in an empirically-derived formula to calculate the IVIS for each treatment group as follows:
IVIS = mean opacity value + (15 x mean permeability OD490 value)
The standard deviation for each group was calculated as well.


DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
1_Test substance
Value:
-0.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
2_Test substance
Value:
-0.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
3_Test substance
Value:
-0.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein leakage
Run / experiment:
1_Test material
Value:
-0.021
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein leakage
Run / experiment:
2_Test material
Value:
-0.008
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein leakage
Run / experiment:
3_Test material
Value:
0.01
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
1_Test material
Value:
-1.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
2_Test material
Value:
-0.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
3_Test material
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The Opacity value of the test substance was -0.6, 15 x Permeability value was -0.090 which resulted in an IVIS of -0.6.
The mean opacity and the mean permeability of the corneas, treated with deionised water (NC) were -1.0 and 0.000 respectively, which are less than the established upper limits for background opacity and permeability values of the historical data of the negative controls.
The mean IVIS of the corneas, treated with 1 % sodium hydroxide (PC) was 123.5, which is within the range of the historical data.

All untreated eyes ("control eyes") were normal at any observation time.

Any other information on results incl. tables

Table1: Opacity, permeability and IVIS values

Opacity, permeability (1 x value measured and 15 x values for IVIS calculation) and IVIS values of test substance, negative and positive controls. Individual data, means and standard deviations (SD).

Substance

Opacity

Permeability (1x)

Permeability (15x)

IVIS

Individual

Mean

SD

Individual

Mean

SD

Mean

SD

Individual

Mean

SD

Aqua dest.
(NC)

-1.2

-1.0

0.4

0.010

0.000

0.010

0.000

0.146

-1.0

-1.0

0.3

-0.6

-0.009

-0.7

-1.3

-0.001

-1.4

1 % Sodium hydroxide
(PC)

91.3

83.9

7.9

2.347

2.642

0.272

39.63

4.082

126.5

123.5

4.2

75.5

2.884

118.8

84.8

2.694

125.2

Test substance

-0.8

-0.6

0.4

-0.021

-0.006

0.015

-0.090

0.231

-1.1

-0.6

0.6

-0.7

-0.008

-0.9

-0.1

0.010

0.0

Unforeseen events

The temperature of the incubator was 29.3 °C – 33.7 °C instead of 31 °C – 33 °C. This deviation is considered to be of no relevance for the outcome of this study.

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
EU GHS
Conclusions:
According to the results of this study and the OECD Guideline 437, the test substance is considered to be not an ocular corrosive or severe irritant.
Executive summary:

The Bovine Corneal Opacity and Permeability Study (BCOP Test Method) was performed to reveal possible ocular corrosivity and severe irritation of the test substance, according to the OECD Guideline 437 for the testing of chemicals “Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants”,.

Fresh isolated and quality checked corneas were mounted in cornea holders and the initial opacity was determined. After equilibration 750 µl of the test substance (as is) were topicallyadministered to 3 isolated bovine corneas to the epithelial surfaces for 10 minutes. After a post-incubation period of 2 hours, the final opacity was measured. Then 1 ml of a fluorescein solution was added on the epithelial site and permeability was measured after 90 minutes.

Two groups of 3 corneas each served as positive and negative controls. Both control substances were administered under identical conditions as the test substance. The following solutions served as control substances:

  • Negative control:       sterile aqua dest.
  • Positive control:        1 % sodium hydroxide.

Finally the IVIS (In Vitro Irritancy Score) was calculated as follows:

IVIS = mean opacity value + (15 x mean permeability).

The opacity and mean permeability values were corrected for background opacity and the negative control permeability values. The mean opacity value results from subtraction of final opacity from initial opacity. A substance that induces an IVIS≥55.1 is defined as ocular corrosive or severe irritant.

The IVIS for the test substance was -0.6.

IVIS of the negative control was -1.0 and for the positive control 123.5, thus demonstrating the validity of the experiment.

According to the results of this study and the OECD Guideline 437, the test substance is considered to be not an ocular corrosive or severe irritant.

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