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Skin irritation / corrosion

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Administrative data

skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
according to guideline
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Specific details on test material used for the study:
Supplier: The Union Carbide/Dow Corporation, Taft, LA (Batch#: TA0355R3JJ)
Purity/Characterization (Method of Analysis and Reference): The purity of Piperazine 68% AQ was determined to be 67.8% (Certificate of Analysis # 2399100, The Dow Chemical Company, Taft, LA, 3/04/2005).
Appearance (Physical State, Color): Colorless liquid

In vitro test system

Justification for test system used:
The EpiDermTM test is based on the experience that corrosive chemicals show cytotoxic effects following short term exposure to the skin. It is designed to predict and classify the skin corrosion potential of a test substance by assessment of its effect
on a three-dimensional human epidermis model. The test involves topical application of the test material to the surface of the EpiDermTM tissue for 3 or 60 minutes followed immediately by determination of cytotoxicity. Cytotoxicity is expressed as
the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3— (4,5— dimethylthiazol—Z-yl)-2,5—diphenyl tetrazolium bromide MTT assay) at the end of the treatment.
other: Milli-Q water
Details on test system:
EpiDermTM Skin Model (EPI— 200). The model consisted of normal human-derived epidermal kerotinoc ytes that were cultured to form a multilayered, highly differentiated model ofthe human epidermis. It consisted of organized basal, spinous and granular
layers, and a multilayered stratum comeum containing intercellular lamellar lipid layers arranged inpattems analogous to those found in vivo. The EpiDerrnTM tissues were cultured on polycarbonate membranes of 10 mm cell culture inserts.
Supplier and Location: MatTek Corporation; Ashland MA, USA.
Amount/concentration applied:
The test material was prepared by further diluting Piperazine AQ68% with Milli-Q® water to obtain 34%, 30%, 25%, 20%, 16%, and 11% solutions. The pH values of all these piperazine solutions were measured using a Denver Basic pH meter (Denver
Instrument Co., Arvada, Colorado).
Duration of treatment / exposure:
The test material was administered by topical application to the skin tissue disc.
The skin tissues were kept in a refrigerator the day they were received. The next day, at least one hour before the assay was started, the tissues were transferred to 6-well plates containing 0.9 ml Dulbecco’s Modified Eagle’s (DMEM; GIBCO, Grand
Island, NY) medium per well. The medium was replaced with fresh DMEM medium just before test compound was applied. Fifty ul of each test solution was added into the 6—well plates on top of the skin tissues. The remaining tissues were treated with 50 ul Milli—Q® water (negative control) or 50 ul 8N KOH (positive control). After the exposure period, the tissues were carefully washed with phosphate buffered saline (PBS; GIBCO, Grard Island, NY) to remove residual test substance. Rinsed tissues
were kept in 24 well plates on 300 ul DMEM medium and placed into a humidified (37°C, 5% C02) incubator until all 12 tissues (= one application time, including controls) were dosed for the particular period and rinsed.
Duration of post-treatment incubation (if applicable):
The DMEM medium was replaced by 300 ul MTT-medium and tissues were incubated for 3 hr at 37°C, 5% C02. After incubation, the tissues were washed with PBS and indicator formazan were extracted with 2 ml isopropanol over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 540 nm in triplicate with the VMax® Microplate Reader (Molecular Devices).
Number of replicates:
The test was performed on a total of 4 tissues per test substance, together with a negative control and a positive control. Two tissues were used for a 3 minute exposure to the test compound, and two tissues were used for a 60 minutes exposure.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
34% - 3 min
Vehicle controls validity:
Positive controls validity:
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
34% - 60 min
Vehicle controls validity:
Positive controls validity:
Remarks on result:
other: corrosive
Other effects / acceptance of results:
Results ofthe assays are summarized in Table 1 (attached). The pH values of 11%, 16%, 20%, 25%, 30% and 34% piperazine test solutions are listed in Table 2.
Mean tissue Viabilities following the three minute exposure period were 97.4%, 99.4%, 94.5%, 88.8%, 103%, 87.1% and following the 60 minute exposure period were 103.8%, 91.2%, 82.8%, 98.7%, 41.4%, 10.2% for the 11%, 16%, 20%, 25%,
30% and 34% piperazine dosing solutions, respectively. Proper conduct of the assay was confirmed as the corrosive 8N potassium hydroxide control samples gave mean tissue viability value of 11.7% at 3 minutes and 6.7% at 60 minutes. Based on the
EpiDermTM prediction model for corrosion, piperazine at 34% was cla ssified as corrosive, while the other dilutions were not classified as corrosive.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (corrosive) based on GHS criteria
Based on the EpiDermTM prediction model for corrosion, piperazine at a 34% concentration was classified as corrosive while all the other dilutions <= 30% were classified as non-corrosive.
Executive summary:

Piperazine was evaluated for skin corrosive potential in an in vitro corrosion assay, EpiDermTM (MatTek Corporation; Ashland Massachusetts), utilizing cultured human epidermal cells. In this assay, test materials are topically applied to three-dimensional regenerated human epidermis tissue for 3 or 60 minutes. Tissue viability is measured using a standard cytotoxicity assay and reported as a percentage of the mean of appropriate negative controls. Skin corrosion potential ofthe test substance is classified according to tissue viability following the two exposure timds. Test substances are considered corrosive ifthe mean viability is less than 50% at 3 minutes or >= 50% at 3 minutes but less than 15% at 60 minutes. In the present study, aqueous solutions of 11%, 16%, 20%, 25%, 30% and 34% piperazine were evaluated. Water served as a negative control and 8N potassium hydroxide was used as a positive control.

The mean viability of tissues treated with 11—30% aqueous solutions ofpiperizine were above 50% at 3 minutes exposure time and above 15% at 60 minutes exposure time. The mean viability ofthe 34% piperizine dose group at 3 minutes and 60 minutes was 87.1% and 10.2 %, respectively. Based on the EpiDermTM prediction model for corrosion, piperazine at a 34% concentration was classified as corrosive while all the other dilutions <= 30% were classified as non-corrosive.