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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1,1-dichloroethylene
EC Number:
200-864-0
EC Name:
1,1-dichloroethylene
Cas Number:
75-35-4
Molecular formula:
C2H2Cl2
IUPAC Name:
1,1-dichloroethene
Details on test material:
Internal test item number: S 10964 11
Batch-No.: 0B 135
Production Date: February 05, 2010
Purity: > 99%
Stability in Solvent: Not relevant
Storage: At room temperature, protected from light, in N2 atmosphere,
original container
Expiration Date: February 04, 2011

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EPISKIN model has been validated for corrosivity testing in an international validation study and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EST-1000 Kit from CellSystems® Biotechnologievertrieb GmbH (53562 St. Katharinen; Germany). The EST-1000 tissue consisted of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consisted 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 EST-1000 tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm ∅).
- Tissue batch number(s): Lot No.: EST 100214-001
- Shipping date: 2 March 2010
- Date of initiation of testing: 4 March 2010

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: At least one hour before starting the assay, tissues were transferred to 6-well plates with assay medium, which is immediately replaced before the test is started. At least one hour before dosing the EST-1000 tissues were removed from the refrigerator. Under sterile conditions using sterile forceps, the inserts were transferred into 6-well plates containing the pre-warmed assay medium. Two 24-well plates were prepared as holding plates, each well containing 300 μL assay medium per well. The holding plates were pre-warmed in an incubator (37 ± 1.5°C, 5 ± 0.5% CO2) until use. After the pre-incubation of the EST-1000 tissues was completed (1 hour 20 minutes for the 1 hour exposure and 1 hour 55 minutes for the 3 minutes exposure) the medium in each well was replaced by 1.0 mL fresh assay medium per well. The negative control (50 µL deionised water) was added to the surface of duplicate EST-1000 tissues. Subsequently, the remaining tissues were exposed to the test item and the positive control in the same manner. The 6-well plates were then placed into an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2).
- Temperature of post-treatment incubation (if applicable): Not applicable

REMOVAL OF TEST MATERIAL AND CONTROLS
At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed using a wash bottle containing PBS to remove any residual test material. Excess PBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper. The tissues were placed in the prepared holding plate until all tissues were rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
The MTT concentrate was prepared freshly and diluted with the MTT diluent. A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well. After the exposure procedure was completed the cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was aspirated from the wells and the wells were rinsed three times with PBS. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol) into each vial. The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted for about 70 hours in the refrigerator.
Per each tissue sample 2 × 200 μL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany) at 570 nm without reference filter. Mean values were calculated from the 2 wells per tissue sample.

NUMBER OF REPLICATE TISSUES: 2

PREDICTION MODEL / DECISION CRITERIA
The mean OD value obtained for the duplicate tissues per test item were used to calculate the percent viability relative to the negative control, which was arbitrarily set at 100%. The test item is considered to be corrosive to skin:
(1) if the viability after 3 minutes exposure is less than 50%, or
(2) if the viability after 3 minutes exposure is greater or equal than 50% and the viability after 1 hour exposure is less than 15%.
 
The test item is considered to be non-corrosive to skin:
(1) if the viability after 3 minutes exposure is greater or equal than 50% and the viability after 1 hour exposure is greater than or equal to 15%.
 
Acceptability of the Assay
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 met the acceptance criterion if the mean OD570 of the two tissues was≥0.8. The assay met the acceptance criterion if mean relative tissue viability of the positive control was ≤ 30%.
Control samples:
other: vehicle control (deionised water, reference) and positive control (KOH 8N)
Amount/concentration applied:
A volume of 50 μL of the undiluted test item was applied to each duplicate tissue.
For the positive and negative controls, a volume of 50 μL was dosed per tissue.
Duration of treatment / exposure:
3 minutes and 1 hour
Number of replicates:
Duplicate

Test system

Details on study design:
The In vitro Skin Corrosion: Human Skin Model Test is based on the observation that skin corrosion (necrotic damage of viable skin cells) shows a high correlation with skin cell cytotoxicity, occurring rapidly after brief exposure of the skin barrier (stratum corneum) to a corrosive chemical. It is designed to predict and classify the skin corrosivity potential of a chemical by using a three-dimensional human epidermis model. The epidermis model (e.g. EST-1000) is derived from human keratinocytes and consists of normal, humanderived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK, which are cultured on specially prepared cell culture inserts using serum free medium, attain levels of differentiation at the cutting edge of in vitro skin technology. Ultrastructurally, the skin models closely parallel human skin. The In vitro Skin Corrosion: Human Skin Model Test consists of topical application of the test material to the tissue for 3 minutes and 1 hour, followed by immediate determination of the cytotoxic effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the exposure period.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: other: Relative absorbance (% of negative control)
Run / experiment:
3 minutes
Value:
79
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: other: Relative absorbance (% of negative control)
Run / experiment:
1 hour
Value:
53
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
After exposure to the test item 1,1-dichloroethylene the relative absorbance values were decreased to 79.0% after 3 minutes exposure. After the 1 hour exposure relative absorbance values were reduced to 53.0%. Nevertheless, both values did not exceed the threshold for corrosivity of 50% (3 minutes) or 15% (1 hour), respectively.

Any other information on results incl. tables

Results:

 Dose group  Exposure Interval  Absorbance 570 nm Tissue 1*  Absorbance 570 nm Tissue 2*  Mean Absorbance of 2 Tissues  Rel. Absorbance
[% of Negative Control]** 
 Negative Control   3 min  1.143 1.391 1.267 100
 Positive Control   3 min  0.438 0.406 0.422 33.3
 1,1-dichloroethylene   3 min  0.991 1.011 1.001 79
 Negative Control   1 hour  1.261 1.193 1.227 100
 Positive Control   1 hour  0.051 0.04 0.046 3.7
 1,1-dichloroethylene   1 hour  0.641 0.66 0.65 53

* Mean of three replicate wells after blank correction

** relative absorbance (rounded values): (100 *(absorbance test item)/absorbance negative control

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the reported experimental conditions, the test item 1,1-dichloroethylene was non corrosive to skin.
Executive summary:

A in vitro study was performed to assess the corrosive potential of 1,1-dichloroethylene by means of the Human Skin Model Test. Independent duplicate tissues of the human skin model EST-1000 were exposed to either the test item, the negative control or the positive control for 3 minutes and 1 hour, respectively. The liquid test item (50 μL) was applied to each tissue and spread evenly over the surface of the tissue. A volume of 50 μL of either the negative control (deionised water) or the positive control (8.0 N KOH) was applied to each tissue. After exposure to the negative control the absorbance values exceeded the required acceptability criterion of mean OD5700.8 for both treatment intervals thereby confirming the acceptable quality of the tissues. Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period and for the 1 hour exposure period thus confirming the validity of the test system. After exposure to the test item 1,1-dichloroethylene the relative absorbance values were decreased to 79.0% after 3 minutes exposure. After the 1 hour exposure relative absorbance values were reduced to 53.0%. Nevertheless, both values did not exceed the threshold for corrosivity of 50% (3 minutes) or 15% (1 hour), respectively. Therefore, the test item was not considered to be corrosive.

In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item 1,1-dichloroethylene was non corrosive to skin.