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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:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented, according to accepted guidelines.

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 draft proposal for a new guideline, In Vitro Skin Irritation - reliability scoring based on 2009 guideline
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Diethyl ether
- Physical state: Liquid (clear)
- Analytical purity: 99.93%
- Lot/batch No.: 09023198
- Expiration date of the lot/batch: 09/2010
- Stability under test conditions: Not reported
- Storage condition of test material: In a closed vessel at room temperature 20 ± 5 °C

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: reconstructed epidermis model
Vehicle:
unchanged (no vehicle)
Control samples:
yes, concurrent negative control
Amount/concentration applied:
30 µL
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
incubation for 42 hours and 47 minutes
Number of replicates:
3

Test animals

Species:
other: Adult human-derived epidermal keratinocytes (Epi-200 tissues)
Strain:
other: not applicable
Details on test animals and environmental conditions:
TEST ANIMALS
-Not applicable

ENVIRONMENTAL CONDITIONS
- Not applicable

Test system

Type of coverage:
other: None; test article was applied topically to tissues (in vitro), which were then covered with a nylon mesh to ensure sufficient contact
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: positive and negative control groups were provided
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
Duration of treatment / exposure:
Single exposure for 60 minutes (of which 35 minutes was at 37 °C)
Observation period:
Not applicable. See details under study design.
Number of animals:
Not applicable.
Details on study design:
This test consists of a topical exposure of a test article to a human reconstructed epidermis model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of 3-[4,5-dimethyl thiazole 2-yl]2,5-diphenyl-tetrazoliumbromide (MTT), present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict skin irritation potential.

Pre-test:
The test article was tested for the ability of direct formazan reduction. The test article was added to MTT reagent and the mixture was incubated in the dark at room temperature for 60 minutes. Untreated MTT reagent was used as the control.

Main test:
The pre-incubated tissues were placed into wells containing assay medium. One plate (3 tissues) was used as the negative control and was treated with phosphate buffered saline. One plate was used as the positive control and was treated with sodium dodecylsulphate and another plate was used for treatment with the test article diethyl ether (a nylon mesh was added in order to ensure sufficient contact with the tissue surface). After an exposure period of 60 minutes (of which 35 minutes was at an incubation temperature of 37 °C), the tissues were rinsed and incubated with assay medium for 42 hours and 47 minutes. The tissues were then transferred into another 24-well plate and incubated with MTT reagent for approximately 3 hours. MTT reagent was then aspirated and replaced with phosphate buffered saline buffer and isopropanol was added. On the next day, formazan was extracted from the wells (2 replicates per well) and measured by determining the optical density at 570 nm.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of three experiments
Value:
112.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: values as percent absorbance; positive control was 10.6%
Other effects / acceptance of results:
Pre-test:
The MTT reagent didn’t change its color within 60 minutes, therefore, direct MTT reduction did not occur and no data correction was necessary.

Main test:
After the treatment, the relative absorbance values were increased to 112.4%. This value is well above the threshold for irritation (50%). Therefore, the test item is considered as not irritant.

Any other information on results incl. tables

As blank, the optical density of isopropanol was measured in 4 wells and the mean absorption value was determined as 0.43. From the measured absorptions for the negative control, positive control, and test article, the mean of each tissue was calculated, subtracting the mean absorption of isopropanol. The mean absorption values for the negative control, test article, and positive control were 2.005, 2.2253, and 0.213, respectively. For the test article and the positive control, the following percentage values for formazan production were calculated in comparison to the negative control, as shown in Table 2. The authors noted that the calculated value was more than 100% because the formazan production in the irritation test was higher than the negative control.

Table 2: % of Formazan Production

Designation

Diethyl Ether

Positive Control

% Formazan production (tissue 1)

105.8

10.3

% Formazan production (tissue 2)

113.5

11.1

% Formazan production (tissue 3)

117.7

10.5

% Formazan production (mean)

112.4

10.6

Validity of the results are shown in Table 3.

Table 3: Validity

Criterion

Demanded

Found

Optical density of negative control

Between 1.0 and 2.5

2.005

% Formazan production of positive control

≤ 20% of negative control

10.6%

Variation within replicates

< 18%

3.9% (negative control)

3.6% (positive control)

5.4% (test item)

The values for the positive control were within the range of historical data of the test facility; however, the value for negative control was not within the range of historical data of the test facility. As the deviation was only 0.4% above the respective range of the historical data, the authors considered this result as uncritical. Variation of biological systems within this order of magnitude is not unusual and furthermore, different cell donors may have been used in the preparation of different batches of the cell cultures.

Applicant's summary and conclusion

Interpretation of results:
other: not classified
Remarks:
Criteria used for interpretation of results: other: CLP (EC 1272/2008)