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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:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according current OECD draft guideline and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
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. Available at http://www.oecd.org.
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tetrahydrolinalool
- Analytical purity: 99%
- Physical state: liquid
- Lot/batch No.: 31047968EO
- Stability under test conditions: stability under storage conditions over the study period
was guaranteed
- pH-value: approx. 5 (undiluted test substance)

Test animals

Species:
other: not applicable
Strain:
other: 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 (incubations with SDS and PBS were performed as negative and positive controls)
Amount / concentration applied:
Thirty microliter of the undiluted liquid test substance was applied using a pipette. A
nylon mesh was placed carefully onto the tissue surface afterwards.
Duration of treatment / exposure:
1 hour
Observation period:
42 hours
Number of animals:
not applicable (3 tissues per treatment group)
Details on study design:
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 positive (5% (w/v) sodium dodecyl sulfate in water) and negative (PBS) controls , respectively.
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 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 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.

Data evaluation:
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.
The quantification of tissue viability is presented as the quotient of the mean OD570 divided by the respective OD570 negative control value in percent.

Acceptance criteria:
Negative control: 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.
Positive control: A viability of ≤ 20% is acceptable.
Tissue variability: The intertissue variability (n=3 tissues) is considered to be acceptable if the SD of %-viability is ≤ 20.

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

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
of negative control
Run / experiment:
mean n=3 tissues
Value:
9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation

Any other information on results incl. tables

mean OD570  Viability (% of NC)  
mean (n=3 tissues) mean (n=3 tissues) SD
negative control 2.0168 100 4.64
test substance 0.1795 9 0.71
positive control 0.1313 7 0.54

Applicant's summary and conclusion