Registration Dossier

Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
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. Available at http://www.oecd.org
Principles of method if other than guideline:
In addition the studies follow the test strategy for determination of a corrosive/irritant property
as given in the following guideline:
- OECD Guideline for Testing of Chemicals No. 404, April 24, 2002 (“Acute Dermal Irritation/Corrosion”)
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of the test substance used in the study report: 2-Chlor-1-(3-hydroxyphenyl)-ethanol
- pH-value: ca. 4.5 (undiluted test substance, moistened with water)
- Homogeneity: The test substance was homogeneous by visual inspection.

Test animals

Species:
other: EpiDerm™ tissues (reconstructed three dimensional human epidermis model)

Test system

Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
Single topical application of 25 µL bulk volume (about 19 mg)
Duration of treatment / exposure:
Two (corrosion test) or three (irritation test) EpiDerm tissue samples were incubated with the test substance for 3 minutes and 1 hour (corrosion test) or 1 hour with about 42 hours post-incubation (irritation test).
Details on study design:
METHOD
Direct MTT reduction: Approximately 25 μL bulk volume of the test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37°C for 55 to 65 minutes. A negative control (50 μL of highly deionized water) was tested concurrently. If the MTT solution color or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT. The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results.

Corrosion test: From the day of arrival in the laboratory, tissues were kept in the refrigerator. At least 1 hour but not more than 1.5 hours before test-substance application, tissues were transferred to 6- well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. The preincubation medium was replaced with fresh medium immediately before application. Two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator, as a rule) and test group (test material, negative control and positive control; 12 tissues per test) were used. For the solid test substance, 25 μL highly deionized water was applied first. Thereafter, a bulk volume of 25 μL of the test material was applied with a sharp spoon and homogeneously distributed with the water. Control tissues were concurrently applied with 50 μL of highly deionized water (negative control, NC) or with 50 μL of 8 n potassium hydroxide (positive control, PC). The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment. Rinsed tissues were kept in 24-well plates (holding plates) at room temperature on assay medium until all tissues per application time were dosed and rinsed. The assay medium was then replaced by MTT solution and tissues were incubated for 3 hours. After incubation, tissues were washed with PBS and the formazan produced by the tissues was extracted with isopropanol over night at room temperature. 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.

Irritation test: On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours. Three tissues were treated with the test substance, the PC and NC, respectively. For the solid test substance, 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 were concurrently applied with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5% SDS (positive control, PC). A nylon mesh was placed carefully onto the tissue surface afterwards. 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. When all tissues were 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 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. After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 h ± 3 min. 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 for at least 2 hours at room temperature on a plate shaker. 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.

Tissue viability: The quantification of tissue viability is presented as the quotient of the mean OD570 divided by the respective OD570 NC value in percent for each exposure time.

ACCEPTANCE CRITERIA:
- Assay acceptance criterion for the negative control (NC): The absolute OD570 of the negative control tissues in the MTT-test 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.
- Assay acceptance criterion for the positive control (PC): Corrosion test: Potassium hydroxide as 8.0 normal ready made solution is used as positive reference. A 3-minute treatment with 8.0 n KOH usually reveals a mean relative tissue viability of ~20%. An assay is acceptable if mean relative tissue viability of the 3 min positive control is ≤ 30%. Irritation test: 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, 2 tissues (corrosion test) or 3 tissues (irritation test) are treated in parallel. The inter-tissue variability is considered to be acceptable if the difference of the OD570 values of the two tissues is ≤ 0.3 (corrosion test) or if the SD of %-viability is ≤ 20 (irritation test).

EVALUATION OF RESULTS:
- Corrosive potential of the test materials is predicted from the mean relative tissue viabilities obtained after 3 min treatment compared to the negative control tissues concurrently treated with highly deionized water. A chemical is considered as "corrosive", if the mean relative tissue viability after 3 min treatment with a test material is decreased below 50%. In addition, those materials with a viability of ≥ 50% after 3 min treatment are considered as "corrosive" if the mean relative tissue viability after 1 hour treatment with a test material is decreased below 15%.
- Irritant potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile PBS. 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 vivo

Resultsopen allclose all
Irritation parameter:
other: viability [% of NC]
Basis:
mean
Score:
103
Remarks on result:
other: Corrosion 3 minutes
Irritation parameter:
other: viability [% of NC]
Basis:
mean
Score:
102
Remarks on result:
other: Corrosion 1 hour
Irritation parameter:
other: viability [% of NC]
Basis:
mean
Score:
99
Remarks on result:
other: Irritation

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information
Executive summary:

In a GLP-compliant, OECD 431 guideline study the potential of the test substance to cause dermal corrosion/irritation was assessed by a single topical application of 25μL bulk volume (about 19 mg) of the test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). Two (corrosion test) or three (irritation test) EpiDerm™ tissue samples were incubated with the test substance for 3 minutes and 1 hour (corrosion test) or 1 hour with about 42 hours post-incubation (irritation test). Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as suitable endpoint. The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 103%, and it was 102% after an exposure period of 1 hour. The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 99%. Based on the observed results it was concluded, that the test substance does not show a skin irritation potential in the EpiDerm skin corrosion / irritation test under the test conditions chosen.