Inorganic residual from kraft or soda pulping separated from green liquor in the chemical recovery cycle.

Eye irritation

Administrative data

Endpoint:

eye irritation

Remarks:

other: Bovine Corneal Opacity and Permeability Study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 May 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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD-Guideline 437, Bovine Corneal Opacity and Permeability Test Method (BCOP), 7th of September 2009.

Deviations:

no

Test guideline 2

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

Test guideline 3

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

Test material information

Constituent 1

Reference substance name:

Inorganic residual from kraft or soda pulping separated from green liquor in the chemical recovery cycle.

EC Number:

923-511-9

IUPAC Name:

Inorganic residual from kraft or soda pulping separated from green liquor in the chemical recovery cycle.

Details on test material:

Name: "Green liquor sludge".Molecular formula: UVCB.Batch No.: Not stated.EC No.: 923-511-9.Appearance: Solid, light grey when dried.Solubility: In water: washed substance is not solubleIn other solvents: not available.pH: Not available.Conditions of storage: Room temperature. Storage in the dark but may be used under light.Stability at conditions of storage: Not available. The test material was not dried.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 Klaus Grandits, Ungerbachstraße 10, A-2860 Kirchschlag.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 controlA solution of 20 % imidazole in deionised water (sterile) was prepared freshly before use. No analyses of the preparation were performed.Preparation of the test substanceThe test substance was administered at a concentration of 20 %. Justification of the solvent is according to the guidelines. No analyses of the test substance and its preparation were performed.Test designBovine 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 4 hours. Then the substances were removed and the corneas were accurately washed and 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 in IVIS was calculated. The positive and negative control groups were simultaneously used for other, concurrently performed studies.Application of test and control substances750 µl test substance preparation 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 4 hours while ensuring that the test substance adequately covered the epithelial surface. Incubation temperature, monitored with a min / max thermometer, was 30.9 °C – 33.5 °C.Post-Exposure IncubationAfter the exposure period substances were removed from the anterior chamber and the epithelium was washed three to six times with EMEM+ to determine the effectiveness of rinsing acidic or alkaline materials and to remove substance residues. 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.Opacity MeasurementOpacity 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, 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 30.9 °C – 33.5 °C.Permeability MeasurementPermeability 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 five concentrations (ranging from 12.5 µg/ml to 0.78 µg/ml) of fluorescein solutions were additionally prepared.OpacityOpacity values were calculated as follows:Opacity value = ax (I0/I) + bI = illuminance (Lux) with the corneaI0 = illuminance (Lux) without cornea a, b = equipment-specific variables The standard deviation for each group was calculated as well.PermeabilityTo ensure that the permeability values measured were in the exponential range the linearity was determined by triple measurement of fluorescein solutions of five concentrations. A linear calibration function and the correlation coefficient thereof was calculated. As the OD490 values of corneas treated with 20 % imidazole lied outside of the linear range dilutions of 1:5 were measured.As the OD490 values of corneas treated with the test substance 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 criteriaThis 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 resultsAccording 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 vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

GLS test substance

Value:

78.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

In vivo

Irritant / corrosive response data:

The Opacity value of "Green liquor sludge" was 14.4, 15 x Permeability value was 64.23 which resulted in an IVIS of 78.6.The IVIS of "Green liquor sludge" was 78.6. A slight increase in opacity and an important increase in permeability was observed.

Other effects:

None

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 Ivalues 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)	6.6	4.9	1.5	0.002	0.000	0.002	0.000	0.03	6.6	4.9	1.5
	3.6			-0.001					3.6
	4.5			-0.001					4.5
20 % Imidazole (PC)	76.4	75.4	0.9	3.385	3.220	0.433	48.3	6.495	127.2	123.7	7.0
	75.0			3.547					128.2
	74.7			2.729					115.7
Test substance	11.3	14.4	7.2	5.582	4.282	1.135	64.23	17.025	95.0	78.6	14.9
	22.6			3.490					75.0
	9.2			3.772					65.8

Unforeseen events

The temperature of the incubator was 30.9 °C – 33.5 °C instead of 31 °C – 33 °C. This deviation is considered to be of no relevance for the outcome of this study.

The positive control IVIS was 123.7. This value is slightly higher than 120.3, which is the upper limit of the mean +/- 2SD of the historical data of the positive control. This deviation is considered to be of no relevance for the outcome of this study.

Applicant's summary and conclusion

Interpretation of results:

corrosive

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Green liquor sludge is regarded to be 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 "Green liquor sludge" needs to be labelled as R41 (EU), Category 1 (EPA and GHS).

Executive summary:

The Bovine Corneal Opacity and Permeability Study (BCOP Test Method) was performed to reveal possible ocular corrosivity and severe irritation of"Green liquor sludge", 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 preparation were topically administered to 3 isolated bovine corneas to the epithelial surfaces for 4 hours andthe 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:        20 % imidazole.

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 "Green liquor sludge" was 78.6.

IVIS of the negative control was 4.9 and for the positive control 123.7, thus demonstrating the validity of the experiment.

According to the results of this study and the OECD Guideline 437, the test substance Green liquor sludge is considered to be an ocular corrosive or severe irritant.

According to the guidelines "Green liquor sludge" needs to be labelled as R41 (EU), Category 1 (EPA and GHS).