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Description of key information

3-Methylpyrazol was found to be corrosive in the Human Skin Model Test.
In the BCOP Test a severe eye irritation potential of 3-Methylpyrazol was determined.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
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
Qualifier:
according to guideline
Guideline:
other: OECD 431
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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.
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.
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)
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.


Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-04-26 - 2011-06-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-study
Qualifier:
according to guideline
Guideline:
other: OECD 437
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
other: bovine eye
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hank’s balanced salt solu-tion (supplemented with 0.01% streptomycin and 0.01% penicillin). Then the corneas were dissected and incubated with media at 32 ± 1°C in an incubation chamber for 1 hour.
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
750 µL
Duration of treatment / exposure:
10 min.
Observation period (in vivo):
120 min
Number of animals or in vitro replicates:
3 bovine eyes
Details on study design:
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32°C ± 1°C.
On the day of the assay, the MEM without Phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32°C ± 1°C.
The same was performed with the MEM with Phenol red but without the sodium bicar-bonate.
After the arrival of the corneas they were examined and only corneas which were free from defects were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM without Phenol red was filled. The holders were then incubated for one hour in the incubation chamber at 32°C.
After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used.
The baseline opacity was measured by placing the holder with the cornea in a spectral photometer and recording the absorption at 570 nm. Opacity is calculated from the meas-ured absorption following the equation stated in chapter “Evaluation”, page 14.
For each treatment group (negative control, positive control and test item), three replicates were used. 750 µl negative control resp. test item resp. positive control solution were ap-plied to each replicate.
According to the characteristics of the test item, the following treatment procedure was performed:
7.3.1 Closed Chamber Method
The “closed chamber-method” is used for non-surface-active liquids. Non-surface-active liquids are used neat.
The respective substance (negative control, positive control or test item) was applied by pipetting 750 µL of the appropriate solution through the refill hole in the holder on the cor-nea. The test item was given on the epithelium in such a manner that as much as possible of the cornea was covered with test item.
750 µL of the test item were tested neat.
Exposition time on the corneas was 10 min at 32°C ± 1°C. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, the anterior chamber was filled with cMEM without phenol red, and the corneas were stored for an additional two hours at 32°C ± 1°C (post-incubation).
After the post-incubation, the cMEM without phenol red was renewed in both chambers. Then, final opacity value of each cornea was recorded (again by measurement at 570 nm). The cMEM without Phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution (concentration 4 mg/ml) was added to the front chamber.
The chambers were then closed again and incubated for 90 min at 32 ± 1 C. After incuba-tion, the content of the posterior chamber was thoroughly mixed. Then, the permeability was measured with the spectral photometer as optical density of the liquid at 490 nm.
Irritation parameter:
other: IVIS (in vitro irritation score)
Basis:
mean
Time point:
other: 10 min
Score:
85.73
Reversibility:
not specified
Interpretation of results:
highly irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
It can be concluded that 3 -Methylpyrazole possesses very severe eye irritation potential.
Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of 3-Methylpyrazolby quantitative measurements of changes in opacity and permeability in a bovine cornea.

The test item 3-Methylpyrazolwas brought onto the cornea of a bovine eye which previously had been incubated with cMEM without Phenol red at 32±1°C for one hour and whose opacity had been determined. The test item was incubated on the cornea for 10 minutes at 32±1°C. After removal of the test item and two hours post-incubation, opacity and permeability values were measured.

Physiological sodium chloride solution was used as negative control, sodium hydroxide (10% solution in demineralised water) was used as positive control.

The positive control induced a very severe irritation on the cornea, mean IVIS was 215.7930.

The negative control showed no irritation, mean IVIS was -0.2160.

The test item was tested pure. A mean IVIS of 85.7370 was calculated, corresponding to a classification as very severely eye irritant.

No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid.

It can be concluded that 3 -Methylpyrazole possesses very severe eye irritation potential.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

For skin- and eye-irritation of 3 -Methylpyrazole 2 reliable in vitro studies are available. 3-Methylpyrazol was found to be corrosive in the Human Skin Model Test. In the BCOP Test a severe eye irritation potential of 3-Methylpyrazol was determined.


Justification for selection of skin irritation / corrosion endpoint:
The study is GLP compliant and has Klimisch score 1.

Justification for selection of eye irritation endpoint:
The study is GLP compliant and has Klimisch score 1.

Effects on skin irritation/corrosion: corrosive

Effects on eye irritation: highly irritating

Justification for classification or non-classification

The criteria for classification as skin corrosiv Cat. 1 B (H314) are fullfilled. In addition 3 -Methylpyrazol has to be classified as eye corrosive substance Cat. 1 (H318).