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Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
in vitro
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 Method11 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:
As the pH determination showed a pH of 5.49, no alkaline / acid reserve was determined.
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. 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.
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 of test material (as cited in study report): "SCP liquor 2".
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

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:
EpiDerm Skin Corrosivity Test
50 μL of the test substance were dispensed directly atop the Epi-200 tissue.
EpiDerm Skin Irritation Test
30 μL of the test substance were dispensed directly atop the Epi-200 tissue.
Duration of treatment / exposure:
EpiDerm Skin Corrosivity Test
Exposure times:
• 3 minutes
• 1 hour
EpiDerm Skin Irritation Test
Exposure time:
• 60 minutes, post-incubation: 42 hours
Observation period:
n.a.
Number of animals:
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.
Epiderm Skin Irritation Test
Three tissue replicates were used, including deionised water as negative and 5 % SDS 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 was 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.

Epiderm Skin Irritation Test
Three tissue replicates were used, including distilled water as negative and 5 % SDS as
positive control.
Exposure time:
• 60 minutes, post-incubation: 42 hours
30 μL of each reference substance were dispensed directly atop the EpiDerm™ tissue.
30 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.
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 using
the extractant solution as blank in a plate spectrophotometer at 570 nm, without reference filter.

Calculations

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 vivo

Resultsopen allclose all
Irritation parameter:
other: Mean tissue viability (%)
Basis:
mean
Time point:
other: 3 minutes
Score:
98.7
Reversibility:
other: n.a.
Remarks on result:
other: Non-Corrosive
Irritation parameter:
other: Mean tissue viability (%)
Basis:
mean
Time point:
other: 1 hour
Score:
102.7
Reversibility:
other: n.a.
Remarks on result:
other: Non-Corrosive
Irritation parameter:
other: Mean tissue viability (%)
Basis:
mean
Time point:
other: 60 minutes, 42 hours post-incubation
Score:
104.5
Reversibility:
other: n.a.
Remarks on result:
other: Non-Irritant
Irritant / corrosive response data:
As the pH determination showed a pH of 5.49, no alkaline / acid reserve was determined.
Epiderm Skin Corrosivity Test:
• The mean percentage viability of the treated skin discs after 3 minutes of exposure was
98.7 % which is above the threshold of 50 % for classification.
• The mean percentage viability of the treated skin discs after 1 hour of exposure was
102.7 % which is above the threshold of 15 % for classification.
Epiderm Skin Irritation Test:
• The mean percentage viability of the treated skin discs was 104.5 % which is above the
threshold 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 (negative
control) was 1.643 after 3 minutes, and 1.545 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 18.9 %, that is lower than
30 %, as required by the assay acceptance criteria.
• The maximum inter tissue viability differences of the "SCP LIQUOR 2" treated skin discs
were 4.8 % for 3 minutes and 8.2 % 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 acceptance
criteria.
• The mean tissue viability of the positive control was 8.1 %, that is lower than 20 %, as
required by the assay acceptance criteria.
• The standard deviation calculated from individual percentual tissue viabilities of the
"SCP LIQUOR 2" treated skin discs was 5.9, that is below 18 % as required by the
assay acceptance criteria.

Applicant's summary and conclusion

Interpretation of results:
not irritating
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
"SCP LIQUOR 2" is considered to be non-corrosive and non-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

"SCP LIQUOR 2".

Firstly the pH was determined:

As the pH determination showed a pH of 5.49, no alkaline / acid reserve was determined.

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

98.7 % which is above the threshold of 50 % for classification.

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

102.7 % which is above the threshold of 15 % for classification.

Epiderm Skin Irritation Test:

• The mean percentage viability of the treated skin discs was 104.5 % which is above the

threshold of 50 % for classification.

Conclusion

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

"SCP LIQUOR 2" is considered to be non-corrosive and non-irritant to skin.