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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Remarks:
In vitro direct topical test and In vitro patch test
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2006
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
In vitro direct topical test and In vitro patch test
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: not specified
Cell source:
other: not specified
Source strain:
not specified
Control samples:
yes, concurrent positive control

Test animals

Species:
other: human tissue; SkinEthic (Nice, France)
Strain:
not specified

Test system

Type of coverage:
open
Preparation of test site:
not specified
Vehicle:
unchanged (no vehicle)
Controls:
yes
Duration of treatment / exposure:
4 h, 37°C
Observation period:
4 h
Details on study design:
In vitro direct topical test protocol:
Three reconstituted epidermal tissues of 0.63cm² on 0.3 ml defined maintenance medium in a 24-well plate were used per control or tested compound. Hundred microliters or 100 mg of test compounds were homogeneously displayed on the total surface of the reconstructed epidermis. Negative controls and positive controls were run in parallel for each experiment. Cultures were incubated for 4h at 37 °C, 5% CO2. The three cultures were then transferred into new wells of the same 24-well plate containing 0.3 ml of maintenance medium. Tissues were washed three times with 0.5 ml saline solution A. With solids (powders or crystals), the insert was turned upside down before washing, and— maintained in this position with forceps-knocked 2- to 3- fold on the inner wall of a beaker to mechanically remove most of the applied compound. Histology, MTT reduction, and IL-1α release endpoints were measured. Untreated tissues and H2O treated tissues were used as negative controls while SDS 20% (Basketter et al., 1997; Fentem et al., 2001) and nonanoic acid (Wahlberg and Maibach, 1980) treated tissues were used as positive controls. Negative controls were considered satisfactory if three criteria were met: a high cell viability measured by MTT reduction (≥85% of untreated epidermis), a normal histology (score≥75) (see histology scoring below) and no release of large amounts of IL-1α (<30pg/mL). Positive controls were considered satisfactory when a low cell viability was measured by MTT reduction (<50%), and when a necrosed histology (score<75) and an increase of the amount of secreted IL-1 α (≥30 pg/mL) were observed.

In vitro patch test protocol:
Three reconstituted epidermal tissues of 4 cm², placed on 1 ml defined maintenance medium in a 6-well plate, were used per control or test compound. Seventy-five microliters of the compound was homogeneously displayed on a 0.95 cm2 polypropylene chamber (Cincinnati, OH, USA), which was immediately applied, carefully, to the center of a 4 cm² culture. In case of solid compounds, 75 mg of the powder or crystals was spread on 0.95 cm² (same surface as for liquids) on the center of the culture, and covered immediately by the above chamber. A 5 mm large brush was used to improve the contact between the compound/patch and the epidermal tissue. The patches were homogeneously applied with delicacy; strong pressure was avoided. Negative controls and positive controls were performed in parallel for each experiment. The chamber was removed after a 4 h incubation at 37 °C, 5% CO2. No washing step was included in this protocol because most liquid compounds were absorbed by the patch. With solids, the culture was turned upside down, and—maintained in this position with forceps-knocked 2- to 3-fold on the inner wall of a beaker to mechanically remove most of the applied compound. Histology, MTT reduction, and IL-1α release endpoints were performed. Untreated tissues and H2O treated tissues were used as negative controls while SDS 20% (Basketter et al., 1997; Fentem et al., 2001) and nonanoic acid (Wahlberg and Maibach, 1980) treated tissues were used as positive controls. Negative controls were considered satisfactory if three criteria were met: a high cell viability measured by MTT reduction (≥85% of untreated epidermis), a normal histology (≥75) and no release of large amounts of IL-1α (<105 pg/mL). Positive controls were considered satisfactory when a low cell viability was measured by MTT reduction (<50%), and when a necrosed histology (<75) and an increase of the amount of secreted IL-1 α (≥105 pg/mL) were observed.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
> 50
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Other effects / acceptance of results:
Direct topical application: irritant: low cell viability, necrosed, amount of IL-1α increase
patch test: irritant: high cell viability, necrosed, amount of IL-1α increase

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria