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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study that is suitable for classification and labelling

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD: Draft Proposal for a New Guideline on In Vitro Skin Irritation: Human Skin Model Test of 06-Jun-2008
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): CAS 41583-09-9, ID 10/0023/1
- Physical state: solid
- Analytical purity: 97.4%
- Impurities (identity and concentrations): 1.3% water
- Lot/batch No.: 00228CN9
- Expiration date of the lot/batch: January 2, 2014
- Stability under test conditions: stable
- Storage condition of test material: room temperature
- Other: The substance was homogenous by visual inspection

Specific details on test material used for the study:
- Lot/batch No.: 00228CN9
- Expiration date of the lot/batch: January 2, 2014
- Stability under test conditions: stable
- Storage condition of test material: room temperature
- Other: The substance was homogenous by visual inspection

Test animals

Species:
human
Strain:
other: not applicable
Details on test animals and environmental conditions:
EPIDERM(TM) 200 kit from MaTek Corp, Ashland MA, USA
reconstructed epidermis, surface 0.6cm2 and cultured in vessels of 1 cm diameter.

Test system

Type of coverage:
open
Preparation of test site:
other: not applicale
Vehicle:
other: no vehicle, moistened with water
Controls:
other: untreated skin tissue
Amount / concentration applied:
25 μl applied to each tissue, spread to match tissue size.
Duration of treatment / exposure:
60 min
Observation period:
48h (recovery period, including 3h MTT incubation)
Number of animals:
not applicable
Details on study design:
To determine whether the test substance is able to reduce MTT directly, the test substance was incubated with the substrate.
Three tissues were treated with each test substance, positive and negative controls, espectively.

25 μL sterile PBS was applied first. Thereafter, a bulk volume of 25 μL of the test material was applied with a sharp spoon and homogeneously distributed together with the fluid.
Control tissues are concurrently applied with 30 μL sterile PBS or 5% SDS (positive control).

The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator. The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. After all tissues had been rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were incubated in the incubator at 37°C for 24 ± 2 hours. After that the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period.

After the postincubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in 2 mL isopropanol for at least 2 hours at room temperature on a plate shaker (ca. 120 rpm). After shaking the isopropanol extract and piercing the tissues, 2 aliquots of each extract per tissue were transferred to a 96-well microtiter plate. The optical density of the extracted formazan complex was determined spectrophotometrically using a filter with a wavelength of 570 nm.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: other: viability of reconstituted epidermis
Value:
90
Remarks on result:
other:
Remarks:
Basis: other: mean of first experiment. Time point: 60 min. Max. score: 100.0. Reversibility: other: not applicable. Remarks: Score given in %. (migrated information)
Irritation / corrosion parameter:
other: other: viability of reconstituted epidermis
Value:
99
Remarks on result:
other:
Remarks:
Basis: other: mean of second experiment. Time point: 60 min. Max. score: 100.0. Reversibility: other: not applicable. Remarks: Score given in %. (migrated information)
Irritation / corrosion parameter:
other: other: Absorbance 570 nm (OD)
Value:
1.973
Remarks on result:
other:
Remarks:
Basis: other: mean of first experiment. Time point: 60 min. Max. score: 2.1808. Reversibility: other: not applicable. (migrated information)
Irritation / corrosion parameter:
other: other: Absorbance 570 nm (OD)
Value:
2.062
Remarks on result:
other:
Remarks:
Basis: other: mean of second experiment. Time point: 60 min. Max. score: 2.0768. Reversibility: other: not applicable. (migrated information)

Any other information on results incl. tables

first experiment   Tissue 1 Tissue 2 Tissue 3 mean SD
Negative control mean OD570 nm 2.2577 2.1342 2.1507 2.1808  
viability [% of negative control] 103.5 97.9 98.6 100 3.07
             
melamine phosphate mean OD570 nm 1.9412 * 2.0047 1.9729  
viability [% of negative control] 89 * 91.9 90 2.06
             
5% (w/v) sodium dodecyl sulfate mean OD570 nm 0.1302 0.1507 0.1487 0.1432  
viability [% of negative control] 6 6.9 6.8 7 0.52
"invalid tissue
second experiment   Tissue 1 Tissue 2 Tissue 3 mean SD
Negative control mean OD570 nm 1.8535 2.3 * 2.0768  
viability [% of negative control] 89.3 110.7 * 100 15.2
             
melamine phosphate mean OD570 nm 2.152 1.924 2.1105 2.0622  
viability [% of negative control] 103.6 92.6 101.6 99 5.85
             
5% (w/v) sodium dodecyl sulfate mean OD570 nm 0.1005 0.114 0.1 0.1048  
viability [% of negative control] 4.8 5.5 4.8 5 0.38

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU