Sulfite liquors and Cooking liquors, white

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2009 - May 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Fully Guideline- and GLP-compliant

Cross-reference

Reason / purpose for cross-reference:

reference to other 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:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD-Guideline 431, "In Vitro Skin Corrosion: Human Skin Model Test ", 13 April 2004

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: DRAFT OECD Guideline “In vitro Skin irritation: Reconstructed Human Epidermis (RhE) Test Method”, Paris 11 December 2009

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ESAC statement, 5 November 2008

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: Regulation (EC) 761/2009: B.46 "In vitro skin irritation: Reconstructed Human Epidermis Model Test". 23 July 2009

Deviations:

no

Principles of method if other than guideline:

Firstly the pH and the alkaline reserve was determined. As the calculation of the alkalinereserve allowed the classification as irritating, no EpiDerm Skin Irritation Test was performed.As the calculation of the alkaline reserve yielded a negative result for classification ascorrosive the test substance was topically applied for 3 minutes and 1 hour to the epidermalsurfaces of three-dimensional human epidermis models, followed by immediatedetermination of the cytotoxic effect.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Sulfite liquors and Cooking liquors, white

EC Number:

268-615-9

EC Name:

Sulfite liquors and Cooking liquors, white

Cas Number:

68131-33-9

Molecular formula:

UVCB substance, not available. View remarks field.

IUPAC Name:

Alkaline pulp cooking substance formed by reacting sodium carbonate in green liquor with calcium hydroxide generated from burnt lime.

Test material form:

liquid

Details on test material:

Name: "WHITE LIQUOR".
Molecular formula: UVCB.
CAS No.: 68131-33-9
Batch No.: Not stated.
Appearance: Yellowish liquid.
Solubility: In water: The substance is a water solution
pH: ~ over 12.
Conditions of storage: Room temperature. Storage in the dark but may be used under light.
Stability at conditions of storage: Not available.
Date of expiry: Not available.

Test animals

Species:

other: in vitro system

Strain:

other: MatTek´s EpiDerm System

Details on test animals or test system and environmental conditions:

MatTek´s EpiDerm System consists of normal, human-derived epidermal keratinocytes whichhave been cultured form a multilayered, highly differentiated model of the human epidermis.It consists of organized basal, spinous and granular layers, and a multi-layered stratumcorneum containing intercellular lamellar lipid layers arranged in patterns analogous to thosefound in vivo. The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially preparedcell culture inserts (MILLICELLs®, 10 mm ∅) and shipped as kits, containing 24 tissues onshipping agarose.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: n.a.

Amount / concentration applied:

50 μL of the test substance mixture were dispensed directly atop the Epi-200 tissue.

Duration of treatment / exposure:

Exposure times:• 3 minutes• 1 hour

Observation period:

n.a.

Number of animals:

Two tissue replicates were used for each treatment (exposure time), including deionisedwater as negative and 8N KOH as positive control.

Details on study design:

Determination of pH and alkaline / acid reservePrior to starting the Epiderm Skin Corrosivity Test the pH-value of a 10 % (w/w) aqueoussolution of the test substance and the alkaline / acid reserve were determined.Substances with a pH < 2.0 or pH > 11.5 and a high buffering capacity need not to be testedfor skin corrosion.Epiderm Skin Corrosivity TestTwo tissue replicates were used for each treatment (exposure time), including deionisedwater as negative and 8N KOH as positive control.Exposure times:• 3 minutes• 1 hour50 μL of each reference substance were dispensed directly atop the EpiDerm™ tissue.50 μL of the test substance were dispensed directly atop the Epi-200 tissue.MTT-testAfter incubation with the test substance and washing with PBS, the tissues were incubatedwith MTT medium at 37°C and 5 % CO2. After 3 hours, the MTT medium was aspirated fromall wells and the tissues were gently rinsed with PBS (2 times). For extraction, the tissueswere incubated with extractant solution (isopropanol) for 2 hours with shaking.After the extraction period, the tissues were pierced with an injection needle and the extract(now a blue formazan solution) was allowed to run into the well from which the tissue wastaken. The 24-well plates were placed on a shaker for 15 minutes until the solutions werehomogeneous in colour.Per each tissue 3 × 200 μL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate (see plate design Figure 1) and the OD was measured usingthe extractant solution as blank in a plate spectrophotometer at 570 nm, without referencefilter.CalculationsDetermination of alkaline reserveThe volume of HCl required to titrate to a pH of 10.00 is measured. The alkaline reserveexpressed as grams of sodium hydroxide in 100 ml of sample is calculated.Cell viabilityCell viability was calculated for each tissue as percent of the mean of the negative controltissues. The skin corrosivity/irritation potential of the test substance was classified accordingto remaining cell viability obtained after test substance treatment with either of the twoexposure times.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: Mean tissue viability (%)

Value:

22.5

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 3 minutes. Reversibility: other: n.a.. Remarks: Corrosive. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: Mean tissue viability (%)

Value:

17.6

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 1 hour. Reversibility: other: n.a.. Remarks: Corrosive. (migrated information)

In vivo

Irritant / corrosive response data:

Determination of pH and alkaline reserve:The pH of a 10 % (w/w) aqueous solution of "WHITE LIQUOR" was 12.67 which is higherthan 11.5, thus the alkaline reserve was determined.The pH + 1/12 alkaline reserve was 13.89 which is below the threshold of 14.5 forclassification as corrosive and the pH + 1/6 alkaline reserve was 15.06 which is above thethreshold of 13 for classification as irritant.Epiderm Skin Corrosivity Test:• The mean percentage viability of the treated skin discs after 3 minutes of exposure was22.5 % which is below the threshold of 50 % for classification.• The mean percentage viability of the treated skin discs after 1 hour of exposure was17.6 % which is above the threshold of 15 % for classification.

Other effects:

Assay acceptance criteria according to the protocol INVITTOX n°119 by ECVAM:• The mean optical density (OD) of the tissues, treated with deionised water (negativecontrol) was 2.239 after 3 minutes, and 2.223 after 1 hour of exposure, that is higher than0.8, as required by the assay acceptance criteria.• The mean tissue viability of the 3 minutes positive control was 27.8 %, that is lower than30 %, as required by the assay acceptance criteria.• The maximum inter tissue viability differences of the "WHITE LIQUOR" treated skin discswere 18.2 % for 3 minutes and 51.5 % for 1 hour exposure, that is below 30 % as requiredby the assay acceptance criteria for the 3 minutes exposure, but above 30 % for the 1hour exposure. However, since the substance was very corrosive already after 3minutes, the skins were completely gone into pieces at the 1 hour timepoint, which is mostprobably the explanation for the variation. Therefore, there is no problem with the validityof the results.

Applicant's summary and conclusion

Interpretation of results:

corrosive

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

According to the results of this study and the Directive 2001/59/EC, the test substance"WHITE LIQUOR" is considered to be corrosive and irritant to skin.

Executive summary:

The Epiderm Skin Corrosivity/Irritation Test (Model EPI-200) was performed to reveal

possible irreversible tissue damages of the skin following the application of

"WHITE LIQUOR".

Firstly the pH and the alkaline reserve was determined:

The pH of a 10 % (w/w) aqueous solution of "WHITE LIQUOR" was 12.67 which is higher

than 11.5, thus the alkaline reserve was determined.

As the calculation of the alkaline reserve allowed the classification as irritating (the pH + 1/6 alkaline reserve was 15.06

which is above the threshold of 13), no EpiDerm Skin Irritation Test was performed.

As the calculation of the alkaline reserve yielded a negative result for classification as

corrosive (pH + 1/12 alkaline reserve was 13.89 which is below the threshold of 14.5)

the test substance was topically applied for 3 minutes and 1 hour to the epidermal

surfaces of three-dimensional human epidermis models, followed by immediate

determination of the cytotoxic effect.

Epiderm Skin Corrosivity Test:

• The mean percentage viability of the treated skin discs after 3 minutes of exposure was

22.5 % which is below the threshold of 50 % for classification.

• The mean percentage viability of the treated skin discs after 1 hour of exposure was

17.6 % which is above the threshold of 15 % for classification.

Conclusion

According to the results of this study and the Directive 2001/59/EC, the test substance

"WHITE LIQUOR" is considered to be corrosive and irritant to skin.