Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

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 Guideline for Testing of Chemicals No. 431, April 13, 2004 (“In vitro Skin Corrosion: Human Skin Model Test”)
GLP compliance:
yes (incl. certificate)
Remarks:
testing lab.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name of the test substance used in the study report: Adamantyldimethylamine
Purity: 99.415 w% (Dimethyladamantylamin)
pH-value: ca. 4 (undiluted test substance)

Test animals

Species:
other: human epidermis model
Strain:
other: human epidermis model
Details on test animals and environmental conditions:
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 EpiDerm tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and commercially available as kits (EpiDerm200), containing 24 tissues on shipping agarose.

Test system

Type of coverage:
other: in vitro test
Preparation of test site:
other: in vitro test
Vehicle:
unchanged (no vehicle)
Controls:
other: not applicable (in vitro test)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µl (corrosion test) or 30 µl (irritation test)
Duration of treatment / exposure:
3 min and 1 h (corrosion test); 1 h followed by a 42-h post-incubation period (irritation test)
Observation period:
not applicable (in vitro test)
Number of animals:
2 tissues (corrosion test); 3 tissues (irritation test)
Details on study design:
Mesh compatibility: For liquid test substances a nylon mesh can be used as a spreading support. However, it was judged that this was not necessary for the test substance. Thus no pretest for mesh compatibility was performed.
Direct MTT reduction: Approximately 50 μL 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 de-ionized 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. In case that direct MTT reduction occurred, one freeze-killed control tissue per exposure time was treated with, each, the test article and the negative control.

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. In addition, 1 killed tissue per exposure time was treated in order to detect direct MTT reduction.
Fifty microliter (50 μL) of the undiluted liquid test substance was applied using a pipette. Control tissues were concurrently applied with 50 μL of highly de-ionized water (negative control, NC) or with 50 μL of 8 n potassium hydroxide (positive control, PC) or test substance (killed tissue control, KC).
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 with 0.9 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. Thirty microliter (30 μL) of the undiluted liquid test substance was applied using a pipette. Control tissues were concurrently applied with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5% SDS (positive control, PC). 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 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 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.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other:
Value:
65
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 3 min. Max. score: 100.0. Reversibility: other: not applicable. Remarks: corrosion test. (migrated information)
Irritation / corrosion parameter:
other:
Value:
89
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 3 min. Max. score: 100.0. Reversibility: other: not applicable. Remarks: corrosion test. (migrated information)
Irritation / corrosion parameter:
other:
Value:
8
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 1 h. Max. score: 100.0. Reversibility: other: not applicable. Remarks: corrosion test. (migrated information)
Irritation / corrosion parameter:
other:
Value:
15
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 1 h. Max. score: 100.0. Reversibility: other: not applicable. Remarks: corrosion test. (migrated information)
Irritation / corrosion parameter:
other:
Value:
7
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 1 h. Max. score: 100.0. Reversibility: other: not applicable. Remarks: irritation test. (migrated information)

In vivo

Irritant / corrosive response data:
The value for inter-tissue variability of the test substance for the exposure period of 3 minutes was again out of the acceptance range. Since all other quality criteria of the test were met and the viability values of both tissues are well above the cut-off for skin corrosion after 3 minute exposure, this deviation is not considered to adversely affect the result of this study.
Overall the results of both runs indicate a corrosion potential of the test substance after the 1-hour exposure period.

Applicant's summary and conclusion

Conclusions:
Based on the observed results and applying the evaluation criteria it was concluded, that Adamantyldimethylamine shows a corrosive potential in the EpiDerm skin corrosion/irritation test under the test conditions chosen.