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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and 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
Guideline:
other: OECD Guideline for Testing of Chemicals No. 431, April 13, 2004 (“In vitro Skin Corrosion: Human Skin Model Test”) and OECD Guideline for Testing of Chemicals No. 439, July 22, 2010 (“In vitro Skin Irritation: Reconstructed Human Epidermis Test Model”)
GLP compliance:
yes (incl. QA statement)
Remarks:
testing lab.

Test material

Constituent 1
Chemical structure
Reference substance name:
Isopulegol
EC Number:
201-940-6
EC Name:
Isopulegol
Cas Number:
89-79-2
Molecular formula:
C10H18O
IUPAC Name:
isopulegol
Details on test material:
- Test substance : Cyclohexanol, 5-methyl-2-(1-methylethenyl)-, (1R,2S,5R)-
- Name of test material (as cited in study report): L-Isopulegol
- Physical state: liquid, colorless clear
- Analytical purity: 99.4 corr.area-%
- Lot/batch No.: Muster 7 CH
- Expiration date of the lot/batch: 01 Jun 2012
- Storage condition of test material: room temperature, under N2
- Other: pH-value approx. 5 (undiluted test substance)

Test animals

Species:
human
Strain:
other: not applicable
Details on test animals or test system and environmental conditions:
not applicable

Test system

Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: not applicable
Amount / concentration applied:
50 µl (corrosion test)
30 µl (irritation test)
Duration of treatment / exposure:
3 min and 1 h
Observation period:
not applicable
Number of animals:
not applicable
Details on study design:
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. The EpiDermTM tissues (surface 0.6 cm²) are commercially available as kits (EpiDerm™ 200).

Direct MTT reduction by the test substance was assessed. 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, in the same way as described in section “Basic procedure”, additionally.

Corrosion test:
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. Fifty microliter (50 µL) of the undiluted liquid test substance was applied using a pipette. Control tissues were concurrently applied with 50 uL of highly de-ionized water (negative control, NC) or with 50 μL of 8-n potassium hydroxide (positive control, PC). A nylon mesh was placed carefully onto the tissue surface of the NC afterwards. The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment. 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:
Three tissues were treated with the test substance, the PC (5% SDS) and NC (sterile PBS), respectively. Thirty microliter (30 µL) of the undiluted liquid test substance, PC and NC was applied using a pipette. 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 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

Any other information on results incl. tables

Corrosion test: The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 120%, and it was 28% after an exposure period of 1 hour.

Irritation test: The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 8%.

The test substance is not able to reduce MTT directly.

Applicant's summary and conclusion

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