1H-Pyrazole-4-carboxylic acid, 3-(difluoromethyl)-1-methyl-, ethyl ester

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, no restrictions, fully adequate for assessment.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline no. 439: In vitro skin irritation: Reconstructed Human Epidermidis Test Method (adopted 22 July 2010)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxylate

EC Number:

619-510-5

Cas Number:

141573-95-7

Molecular formula:

C8H10F2N2O2

IUPAC Name:

ethyl 3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxylate

Details on test material:

- Name of test material (as cited in study report): DFMMP
- Lot/batch No.: Batch No. MKA13111
- Substance type: mono-constituent substance
- Physical state: powder
- Analytical purity: 99.0% (GC)

Test animals

Species:

other: In vitro human skin model

Strain:

other: EPISKIN Standard model

Details on test animals or test system and environmental conditions:

TEST MATERIAL
- Source: SkinEthic Laboratories, Lyon, France
- Acclimation period: On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 24 hours at 37°C.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37.1 – 37.4°C
- Humidity (%): 88 – 89% containing 5 +/- 0.5% CO2 in air
- Photoperiod (hrs dark / hrs light): dark

Test system

Type of coverage:

other: In vitro human skin model

Preparation of test site:

other: In vitro human skin model

Vehicle:

unchanged (no vehicle)

Controls:

other: Negative control: Phosphate buffered saline; positive control: 5% Sodium dodecyl sulphate in PBS.

Amount / concentration applied:

An amount of 11.6 to 14.9 mg of DFMMP was applied directly on top of the skin tissue

Duration of treatment / exposure:

The skin tissues were exposed to DFMMP for 15 minutes at room temperature.

Observation period:

After the exposure period of 15 minutes, the tissues were washed to remove residual test substance, dried and moved to a new well with fresh medium. Subsequently the skin tissues were incubated for 42 hours at 37°C and hereafter determination of the cytotoxic (irritancy) effect was performed.

Number of animals:

Not applicable (in vitro study)

Details on study design:

The objective of this study was to evaluate DFMMP for its ability to induce skin irritation. For this purpose DFMMP was topically applied on a human three dimensional epidermal model and hereafter the cytotoxic (irritancy) effect was determined. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

PROTOCOL
- DFMMP was checked for possible direct MTT reduction before the study was started.
- The test was performed on a total of 3 tissues per treatment (DFMMP, negative and positive control). The tissues were exposed for 15 minutes.
- After a 42 hour post-incubation period, an extraction of formazan was performed and the amount of extracted formazan was determined spectrophotometrically at 570 nm
- Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.

INTERPRETATION OF RESULTS
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should be within the laboratory historical control data range (SD % viability should be 18).
b) The mean relative tissue viability of the positive control should be 40% relative to the negative control (SD % viability should be 18).
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be 18.

A test substance is considered irritant in the skin irritation test if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test is ≤ 50% of the mean viability of the negative controls. A test substance is considered non-irritant in the in vitro skin irritation test if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance is > 50% of the mean viability of the negative controls.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: Relative mean tissue viability compared to the negative control

Remarks on result:

other:

Remarks:

Basis: mean. Remarks: The relative mean tissue viability after 15 minutes treatment with DFMMP compared to the negative control tissues was 101%.. (migrated information)

In vivo

Irritant / corrosive response data:

The relative mean tissue viability obtained after 15 minutes treatment with DFMMP compared to the negative control tissues was 101%. Since the mean relative tissue viability for DFMMP was above 50% after 15 minutes treatment DFMMP is considered to be non-irritant.