Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 April to 28 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Study

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:
other: OECD-Guideline 437, Bovine Corneal Opacity and Permeability Test Method (BCOP), 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
Reference substance name:
Not yet assigned
IUPAC Name:
Not yet assigned
Constituent 2
Reference substance name:
Spent liquor from semi-chemical pulping containing spent inorganic process chemicals and dissolved organic substances originating from the wood raw material
IUPAC Name:
Spent liquor from semi-chemical pulping containing spent inorganic process chemicals and dissolved organic substances originating from the wood raw material
Details on test material:
Name: "SCP liquor 1".
Chemical name: Spent liquor from semi-chemical pulping.
Molecular formula: UVCB.
Batch No.: Not stated.
CAS No.: 66071-92-9
EC No.: 266-111-3.
Appearance: Brown and viscous liquid
Solubility: In water: The substance is a water solution.
pH: ~ 7.
Conditions of storage: Room temperature. Storage in the dark but may be used under light.
Stability at conditions of storage: Not available.
Expiry date: Not available.

Test animals / tissue source

Species:
other: Isolated corneas from the eyes of cows and bulls
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Corneas: Isolated corneas from the eyes of cows and bulls aged between 18 – 20 month and free of macroscopically visible defects.
Supplier: Slaughterhouse Kloiber, Neidlinger Straße 1, A-3121 Karlstetten.
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)
Amount / concentration applied:
750 µL per cornea
Details on study design:
Preparation of the positive control
A solution of 1 % sodium hydroxide in deionised water (sterile) was prepared freshly before use. No analyses of the preparation were performed.

Preparation of the test substance
The test substance was administered undiluted as received. No analyses of the test substance were performed.

Test design
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.

Application of test and control substances
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-Exposure Incubation
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 and to remove the entire substance. 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.

Opacity Measurement
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.

Application of Fluorescein
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.

Permeability Measurement
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 490 nm. 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.

Opacity
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.

Permeability
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.

IVIS (In Vitro Irritancy Score)
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.

Assay acceptance criteria
This test is considered acceptable if it meets the following criteria:
1. The negative control response results in opacity and permeability values that are less than the upper limits for background opacity and permeability values of the historical data of the negative controls.
2. The IVIS of the positive control is within two standard deviations of the historical positive control mean.
The historical values of the negative and positive control includes all generated data of this year.

Interpretation of results
According to the guidelines, an irritation potential of a test substances is predicted if the mean IVIS of three individual corneas exposed to the test substance is ≥ 55.1.

Results and discussion

In vivo

Irritant / corrosive response data:
The Opacity value of "SCP liquor 1" was 1.0, 15 x Permeability value was 0.09 which resulted in an IVIS of 1.1.
The IVIS of "SCP liquor 1" was 1.1. No increase in opacity or permeability was observed.
Other effects:
None

Any other information on results incl. tables

Table 1: Opacity, permeability and IVIS values

Substance

Opacity

Permeability (1x)

Permeability (15x)

IVIS

Individual

Mean

SD

Individual

Mean

SD

Mean

SD

Individual

Mean

SD

Aqua dest.
(NC)

2.0

1.7

1.1

-0.001

0.000

0.001

0.000

0.015

2.0

1.7

1.1

0.5

-0.001

0.5

2.6

0.001

2.6

1 % Sodium hydroxide
(PC)

75.5

99.9

34.3

2.092

2.165

0.298

32.475

4.47

106.9

132.3

31.6

139.1

1.909

167.8

85.0

2.492

122.4

Test substance

0.2

1.0

1.0

0.001

0.006

0.006

0.09

0.09

0.2

1.1

1.1

0.7

0.007

0.8

2.1

0.012

2.3

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:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
"SCP liquor 1" is regarded to be not an ocular corrosive or severe irritant, 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”.
According to the results of this study and the Directive 2001/59/EC for classification, the test substance "SCP liquor 1" needs not to be labelled as R41 (EU), Category 1 (EPA and GHS).
Executive summary:

TheBovine Corneal Opacity and Permeability Study (BCOP Test Method)was performed to reveal possible ocular corrosivity and severe irritation of"SCP liquor 1", according tothe 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 "SCP liquor 1" was 1.1.

IVIS of the negative control was 1.7 and for the positive control 132.3, thus demonstrating the validity of the experiment.

According to the results of this study and the OECD Guideline 437, the test substance "SCP liquor 1" is considered to be not an ocular corrosive or severe irritant.

According to the guidelines "SCP liquor 1" needs not to be labelled as R41 (EU), Category 1 (EPA and GHS).