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

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 yielded a negative result for classification as corrosive and as irritant the testsubstance was topically applied for 3 minutes and 1 hour to the epidermal surfaces of threedimensionalhuman epidermis models, followed by immediate determination of the cytotoxiceffect. As there was no corrosive effect observed, the test substance was topically applied for60 minutes to the epidermal surfaces of three-dimensional human epidermis models. After apost-incubation of 42 hours, a cell viability test was performed.

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 of test material (as cited in study report): "GREEN LIQUOR SLUDGE".Chemical 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 solublepH: Not available.Conditions of storage: Room temperature. Storage in the dark but may be used under light.Stability at conditions of storage: No data 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:

A flat “cookie like” piece of about 8 mm diameter was formed and placed atop the tissue,wetted with 15 ml H2O.

Duration of treatment / exposure:

Epiderm Skin Corrosivity Test:• 3 minutes• 1 hourEpiderm Skin Irritation Test:• 60 minutes, post-incubation: 42 hours

Observation period:

n.a.

Number of animals:

Epiderm Skin Corrosivity TestTwo tissue replicates were used for each treatment (exposure time), including distilled wateras negative and 8N KOH as positive control.Epiderm Skin Irritation TestThree tissue replicates were used, including distilled water as negative and 5 % SDS aspositive 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.A flat “cookie like” piece of about 8 mm diameter was formed and placed atop the tissue,wetted with 15 ml H2O.Epiderm Skin Irritation TestThree tissue replicates were used, including distilled water as negative and 5 % SDS aspositive control.Exposure time:• 60 minutes, post-incubation: 42 hours30 μL of each reference substance were dispensed directly atop the EpiDerm™ tissue.A flat “cookie like” piece of about 8 mm diameter was formed and placed atop the tissue,wetted with 15 ml H2O.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.For the Epiderm Skin Corrosivity Test per each tissue 3 × 200 μL aliquots, for the Epiderm Skin Irritation Test 2 × 200 μL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate 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:

96.1

Remarks on result:

other:

Remarks:

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

Results 2

Irritation / corrosion parameter:

other: other: Mean tissue viability (%)

Value:

15.3

Remarks on result:

other:

Remarks:

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

Results 3

Irritation / corrosion parameter:

other: other: Mean tissue viability (%)

Value:

8.8

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 60 minutes, 42 hours post-incubation. Reversibility: other: n.a.. Remarks: Irritant, R38. (migrated information)

In vivo

Irritant / corrosive response data:

Determination of pH and alkaline reserve:The pH of a 10 % (w/w) aqueous solution of "GREEN LIQUOR SLUDGE" was 12.88 which ishigher than 11.5, thus the alkaline reserve was determined.The pH + 1/12 alkaline reserve was 12.19 which is below the threshold of 14.5 forclassification as corrosive and the pH + 1/6 alkaline reserve was 12.26 which is below 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 was96.1 % which is above the threshold of 50 % for classification.• The mean percentage viability of the treated skin discs after 1 hour of exposure was15.3 % which is above the threshold of 15 % for classification.Epiderm Skin Irritation Test:• The mean percentage viability of the treated skin discs was 8.8 % which is below thethreshold of 50 % for classification.

Other effects:

Epiderm Skin Corrosivity Test: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 1.990 after 3 minutes, and 2.111 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 24.1 %, that is lower than30 %, as required by the assay acceptance criteria.• The maximum inter tissue viability differences of the "GREEN LIQUOR SLUDGE" treatedskin discs were 4.8 % for 3 minutes and 10.0 % for 1 hour exposure, that is below 30 %as required by the assay acceptance criteria.Epiderm Skin Irritation Test:Assay acceptance criteria according to the protocol used during the ECVAM validation study:• The mean OD of the tissues, treated with deionised water (negative control) was 2.4 ,that is higher than 1.0 and lower than 2.5, as required by the assay acceptancecriteria.• The mean tissue viability of the positive control was 8.1 %, that is lower than 20 %, asrequired by the assay acceptance criteria.• The standard deviation calculated from individual percentual tissue viabilities of the"GREEN LIQUOR SLUDGE" treated skin discs was 1.6, that is below 18 % asrequired by the assay acceptance criteria.

Applicant's summary and conclusion

Interpretation of results:

irritating

Remarks:

Migrated informationCriteria used for interpretation of results: EU

Conclusions:

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

Executive summary:

The assessment of alkaline reserve and the Epiderm Skin Corrosivity/Irritation Test (Model EPI-200) were performed to reveal possible irreversible tissue damages of the skin following the application of "GREEN LIQUOR SLUDGE".

Firstly the pH and the alkaline reserve was determined:

The pH of a 10 % (w/w) aqueous solution of "GREEN LIQUOR SLUDGE" was 12.88 which is higher than 11.5, thus the alkaline reserve was determined.

As the calculation of the alkaline reserve yielded a negative result for classification as corrosive (pH + 1/12 alkaline reserve was 12.19 which is below the threshold of 14.5) and as irritant (the pH + 1/6 alkaline reserve was 12.26 which is below the threshold of 13)

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

three-dimensional human epidermis models, followed by immediate determination of the cytotoxic effect.

As there was no corrosive effect observed, the test substance was topically applied for 60 minutes to the epidermal surfaces of three-dimensional human epidermis models. After a post-incubation of 42 hours, a cell viability test was performed.

Epiderm Skin Corrosivity Test:

• The mean percentage viability of the treated skin discs after 3 minutes of exposure was 96.1 % which is above the threshold of 50 % for classification.

• The mean percentage viability of the treated skin discs after 1 hour of exposure was 15.3 % which is above the threshold of 15 % for classification.

Epiderm Skin Irritation Test:

• The mean percentage viability of the treated skin discs was 8.8 % which is below the threshold of 50 % for classification.

Conclusion

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