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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
Study period:
2011-04-19 - 2011-06-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD 431
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
3-methylpyrazole
EC Number:
215-925-7
EC Name:
3-methylpyrazole
Cas Number:
1453-58-3
Molecular formula:
C4H6N2
IUPAC Name:
3-methyl-1H-pyrazole
Test material form:
other: liquid

Test animals

Species:
other: human skin model
Strain:
other: not applicable
Details on test animals or test system and environmental conditions:
The EpiDermTM tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epi-dermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analo-gous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell cultures inserts.

Test system

Type of coverage:
open
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: deionised water
Amount / concentration applied:
50 µL
Duration of treatment / exposure:
3 min, 1 hour
Number of animals:
2 tissues
Details on study design:
Four 6-well-plates were prepared with 0.9 mL assay medium in each well. The inserts containing the tissues were transferred to the wells using sterile forceps and the 6-well-plates were set into the incubator at 37 oC and 5% CO2 for one hour (pre-incubation).
For each experiment (“three minutes” and “one hour”), one 24-well-plate was prepared as holding plate. 12 wells of each plate were filled with 300 µL assay medium, the other 12 with 300 µL MTT reagent. One additional plate was left empty. The plates were stored in the incubator.
For each experiment (“three minutes” and “one hour”), two 6-well-plates were used. After pre-incubation, the assay medium was replaced by fresh assay medium and the test was started, using two wells as negative control with 50 µL H2O demin., two wells as positive controls with 50 µL potassium hydroxide solution and two other wells for testing the test item.
The liquid test item was applied without preparation (50 µL).
At the start of each experiment (application of negative controls), a stop watch was started.
After the respective incubation time (three minutes ± 10 sec and one hour), the inserts were removed from the plates using sterile forceps. The inserts were thoroughly rinsed with PBS, blotted with sterile cellulose tissue and set into the respective holding plate, using the wells containing assay medium. After transfer of all inserts, they were immediately moved to the wells containing MTT reagent, blotting the bottom with cellulose tissue again before setting the insert into the MTT well. The tissues were incubated with MTT reagent for three hours. After this time, the MTT reagent was aspirated and replaced by PBS buffer. This was then aspirated, too, and replaced several times. At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanole were pipetted, taking care to reach the upper rim of the insert. The plate was then covered with Parafilm® and left to stand over night at room temperature.
On the next day, the inserts in which formazan had been produced over night were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation.
From each well, three replicates with 200 µL solution (each) were pipetted into a 96-well-plate which was read in a plate spectral photometer at 570 nm.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: other: % Formazan production
Value:
73.8
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 3 min. Max. score: 50.0. Reversibility: no data. (migrated information)
Irritation / corrosion parameter:
other: other: % Formazan production
Value:
14.9
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 1 hour. Max. score: 15.0. Reversibility: no data. (migrated information)

Applicant's summary and conclusion

Interpretation of results:
corrosive
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item is considered corrosive.
After three minutes treatment, the relative absorbance values were decreased to 73.8 %. This value is well above the threshold for corrosivity (50 %). After one hour treatment, though, relative absorbance values were reduced to 14.9 %. This value is below the threshold for corrosivity (15 %).
The values of the negative control were well above the required acceptability criterion of mean OD > 0.8 for both treatment intervals thus showing the quality of the tissues.
The value of the positive control induced a decrease in the relative absorbance as com-pared to the negative control to 29.2 % for the three minutes treatment interval and 24.2 % for the one hour treatment interval thus ensuring the validity of the test system.
For these reasons, the result of the test is considered valid.
Executive summary:

One valid experiment was performed.

Two tissues of the human skin model EpiDermTMwere treated with3-Methylpyrazolfor three minutes and one hour, respectively.

50 µL of the liquid test item were applied to each tissue and spread to match the tissue size. Deionised water was used as negative control, 8m KOH was used as positive control.

After treatment with the negative control the absorbance values were well above the required acceptability criterion of mean OD > 0.8 for both treatment intervals thus showing the quality of the tissues. The positive control showed clear corrosive effects for the three minutes treatment.

After three minutes treatment with the test item, the relative absorbance values were reduced to 73.8 %. This value is well above the threshold for corrosion potential (50 %). After one hour treatment, relative absorbance values were reduced to 14.9 %. This value is below the threshold for corrosion potential (15 %).

Therefore,3-Methylpyrazolis considered as corrosive in the Human Skin Model Test.