1H-Pyrazole-5-carboxamide, 4-amino-1-methyl-3-propyl-, hydrochloride (1:?)

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 November 2015 to 20 November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-amino-1-methyl-3-propyl-1H-pyrazole-5-carboxamide hydrochloride

EC Number:

938-723-7

Cas Number:

1688656-88-3

Molecular formula:

C8H14N4O.HCL

IUPAC Name:

4-amino-1-methyl-3-propyl-1H-pyrazole-5-carboxamide hydrochloride

Test material form:

solid: particulate/powder

Details on test material:

Batch 2000533289
Purity/Composition 100.2%

Specific details on test material used for the study:

Batch 2000533289 (taken from label) Purity/Composition 100.2% Test item storage At room temperature Stable under storage conditions until 03 November 2019 (expiry date) (taken from label)

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: MatTek Corporation, Ashland MA, U.S.A.

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

Tissues On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM medium.
DMEM (Dulbecco’s Modified Eagle’s Medium) Supplemented DMEM medium, serum-free supplied by MatTek Corporation and Life Technologies.
MTT medium MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.
Environmental conditions All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 62 - 83%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.1 - 37.3°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Approximately 25 mg of PF-01049573-01; UK-084443-01 was applied directly on top of the skin tissue.

Duration of treatment / exposure:

3-minute and 1-hour treatments

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 MINUTE EXPOSURE

Value:

ca. 82

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 HOUR EXPOSURE

Value:

ca. 88

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

PF-01049573-01; UK-084443-01 was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that PF-01049573-01; UK-084443-01 did not interfere with the MTT endpoint.

The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with PF-01049573-01; UK-084443-01 compared to the negative control tissues was 82% and 88% respectively. Because the mean relative tissue viability for PF-01049573-01; UK-084443-01 was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment PF-01049573-01; UK-084443-01 is considered to be not corrosive.

The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3-minute exposure to the positive control was 6%. The maximum inter-tissue variability in viability between two tissues treated identically was less than 15% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 8%. It was therefore concluded that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Finally, it is concluded that this test is valid and that PF-01049573-01; UK-084443-01 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

In vitro skin corrosion test with  PF-01049573 -01; UK-084443-01 using a human skin model.

This report describes the ability of  PF-01049573-01; UK-084443-01 to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of PF-01049573-01; UK-084443-01 was tested through topical application for 3 minutes and 1 hour.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch 2000533289 of  PF-01049573-01; UK-084443-01 was a white powder with a purity of 100.2%. Skin tissue was moistened with 25 Nl of Milli-Q water and approximately 25 mg of  PF-01049573 -01; UK-084443-01 was applied directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 6% after 3 minutes exposure. The absolute mean OD570  (optical density at 570  nm)  of the negative control tissues was within the laboratory historical control data range. The acceptability criteria for the maximum inter-tissue variability in viability between two tissues treated identically and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues were met, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with  PF-01049573-01; UK-084443-01 compared to the negative control tissues was 82% and 88%, respectively. Because the mean relative tissue viability for  PF-01049573-01; UK-084443-01 was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment  PF-01049573-01; UK-084443-01 is considered to be not corrosive.

Finally, it is concluded that this test is valid and that  PF-01049573-01; UK-084443-01 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.