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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:
2012-04-03 to 2012-07-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant study.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
(2009)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Test animals

Details on test animals and environmental conditions:
Three dimensional human epidermis model
The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, 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 EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.

Test system

Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 30 μL
- Concentration: Undiluted

Duration of treatment / exposure:
The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator.
Observation period:
The tissues were incubated in the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours 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.
Details on study design:
CONTROLS
Negative control (NC): PBS, sterile
Positive control (PC): 5% (w/v) sodium dodecyl sulfate (SDS, Sigma, Germany) in deionized water, sterile
MTT-reduction control (KC): PBS, sterile or test substance

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 isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

Data evaluation
Principle
The OD570 values determined for the various tissues are measures of their viability. The quotient of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether or not a test material is an irritant.

Calculation of individual and mean optical densities
The individual tissue OD570 is calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of three tissues treated in the same way is calculated.

Tissue viability
The quantification of tissue viability is presented as the quotient of the mean OD570 (or mean OD570 KC corrected, if applicable) divided by the respective OD570 NC value in percent.

ACCEPTANCE CRITERIA
In case one of the below given acceptance criteria is not met repetition of the test is considered.
Assay acceptance criterion for the negative control (NC)
The absolute OD570 of the negative control tissues in the MTTtest is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 1.0. The mean OD570 of the NC should not exceed 2.5.

Assay acceptance criterion for the positive control (PC)
5% SDS is used as PC and reflects the sensitivity of the tissues used in the test conditions. A viability of ≤ 20% is acceptable.

Assay acceptance criterion for tissue variability
For every treatment, except the killed controls, 3 tissues are treated in parallel. The inter-tissue variability is considered to be acceptable if the SD of %-viability is ≤ 20.

Assay acceptance criterion for killed controls (KC)
The OD570 of the killed control tissues treated as negative control should be ≤ 0.35.

A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50%.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other:
Value:
114
Remarks on result:
other:
Remarks:
Basis: mean tissue 1-3. Remarks: 1st test run; SD: 4.21. (migrated information)
Irritation / corrosion parameter:
other:
Value:
89
Remarks on result:
other:
Remarks:
Basis: mean tissue 1-3. Remarks: 2nd test run; SD:7.53. (migrated information)

In vivo

Irritant / corrosive response data:
Negative Control: Viability [% of NC]: 100%
Positive Control: Viability [% of NC]
1st run: 4 %
2nd run: 2%

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information