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Toxicological information

Skin irritation / corrosion

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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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD-Guideline 431, "In Vitro Skin Corrosion: Human Skin Model Test ", 13 April 2004
Deviations:
no
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
Qualifier:
according to guideline
Guideline:
other: ESAC statement, 5 November 2008
Deviations:
no
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 alkaline
reserve 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 as
corrosive 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.
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Sulfite liquors and Cooking liquors, green
EC Number:
268-612-2
EC Name:
Sulfite liquors and Cooking liquors, green
Cas Number:
68131-30-6
Molecular formula:
HNa3OS
IUPAC Name:
Aqueous alkaline solution of sodium salts produced by dissolving melt from incineration of spent liquor from sodium based pulping processes.
Details on test material:
Name of test material (as cited in study report):"GREEN LIQUOR 1".
Chemical name: Sulphite liquors and Cooking liquors, green.
Molecular formula: UVCB from sulphite process
Batch No.: Not stated.
CAS No.: 68131-30-6.
EC No.: 268-612-2.
Appearance: Yellowish liquid.
Solubility: In water: The substance is a water solution (82.3 % water).
pH: ~ over 11.
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 which
have 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 stratum
corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those
found in vivo. The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared
cell culture inserts (MILLICELLs®, 10 mm ∅) and shipped as kits, containing 24 tissues on
shipping agarose.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: n.a.
Amount / concentration applied:
50 μL of the test substance 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 deionised
water as negative and 8N KOH as positive control.
Details on study design:
Determination of pH and alkaline / acid reserve
Prior to starting the Epiderm Skin Corrosivity Test the pH-value of a 10 % (w/w) aqueous
solution 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 tested
for skin corrosion.

Epiderm Skin Corrosivity Test
Two tissue replicates were used for each treatment (exposure time), including deionised
water as negative and 8N KOH as positive control.
Exposure times:
• 3 minutes
• 1 hour
50 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-test
After incubation with the test substance and washing with PBS, the tissues were incubated
with MTT medium at 37°C and 5 % CO2. After 3 hours, the MTT medium was aspirated from
all wells and the tissues were gently rinsed with PBS (2 times). For extraction, the tissues
were 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 was
taken. The 24-well plates were placed on a shaker for 15 minutes until the solutions were
homogeneous 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 using
the extractant solution as blank in a plate spectrophotometer at 570 nm, without reference
filter.

Calculations

Determination of alkaline reserve
The volume of HCl required to titrate to a pH of 10.00 is measured. The alkaline reserve
expressed as grams of sodium hydroxide in 100 ml of sample is calculated.

Cell viability
Cell viability was calculated for each tissue as percent of the mean of the negative control
tissues. The skin corrosivity/irritation potential of the test substance was classified according
to remaining cell viability obtained after test substance treatment with either of the two
exposure times.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: other: Mean tissue viability (%)
Value:
29.1
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 3 minutes. Reversibility: other: n.a.. Remarks: Corrosive. (migrated information)
Irritation / corrosion parameter:
other: other: Mean tissue viability (%)
Value:
19.7
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 "GREEN LIQUOR 1" was 12.60 which is higher
than 11.5, thus the alkaline reserve was determined.
The pH + 1/12 alkaline reserve was 12.98 which is below the threshold of 14.5 for
classification as corrosive and the pH + 1/6 alkaline reserve was 13.51 which is above the
threshold of 13 for classification as irritant.
Epiderm Skin Corrosivity Test:
• The mean percentage viability of the treated skin discs after 3 minutes of exposure was
29.1 % which is below the threshold of 50 % for classification.
• The mean percentage viability of the treated skin discs after 1 hour of exposure was
19.7 % 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 (negative
control) was 1.990 after 3 minutes, and 2.111 after 1 hour of exposure, that is higher than
0.8, as required by the assay acceptance criteria.
• The mean tissue viability of the 3 minutes positive control was 24.1 %, that is lower than
30 %, as required by the assay acceptance criteria.
• The maximum inter tissue viability differences of the "GREEN LIQUOR 1" treated skin
discs were 1.8 % for 3 minutes and 2.0 % for 1 hour exposure, that is below 30 % as
required by the assay acceptance criteria.

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
"GREEN LIQUOR 1" is considered to be 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 1".

Firstly the pH and the alkaline reserve was determined:

The pH of a 10 % (w/w) aqueous solution of "GREEN LIQUOR 1" was 12.60 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 13.51

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 12.98 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

29.1 % which is below the threshold of 50 % for classification.

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

19.7 % 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

"GREEN LIQUOR 1" is considered to be corrosive and irritant to skin.