3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxylic acid

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Jun 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

adopted in 2004

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

adopted in 2019

Deviations:

yes

Remarks:

limited details on model (barrier function, morphology, quality control), no check for direct MTT reduction or colour interference, acceptance and evaluation criteria differ from current guideline, no historical control data, no positive control included

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxylic acid

EC Number:

700-093-4

Cas Number:

176969-34-9

Molecular formula:

C6H6F2N2O2

IUPAC Name:

3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxylic acid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: CellSystems, St. Katharinen, Germany

Source strain:

other: EST-1000 reconstructed human epidermis

Details on animal used as source of test system:

Not applicable

Justification for test system used:

Reconstructed human epidermis is the recommended test system for in vitro predicting of a corrosive potential.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EST-1000 (CellSystems, St. Katharinen, Germany)
- Tissue batch number: EST-90427-001
- Date of initiation of testing: 24 Jun 2009

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 min at room temperature and 60 min at 37.0 ± 2 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the incubation period the tissues were carefully washed with PBS.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 2 h
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: A single experiment was performed.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: The skin was moistened with 50 µL 0.9% NaCl and 25 mg of the solid test item were added on top of the skin tissues.

NEGATIVE CONTROL
- Amount applied: 50 µL

Duration of treatment / exposure:

3 and 60 min

Number of replicates:

triplicates for each treatment and control group

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 3 tissues

Run / experiment:

3 min exposure

Value:

94.43

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

The reliability of the test method was confirmed prior to the experimental study by interlaboratory validation.

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 3 tissues

Run / experiment:

60 min exposure

Value:

100.89

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

The reliability of the test method was confirmed prior to the experimental study by interlaboratory validation.

Any other information on results incl. tables

Table 1: Summary of results on skin corrosion

Sample	Exposure time	OD ± SD	% Viability
Test item	3 min	1.68 ± 0.08	94.43
0.9% NaCl	3 min	1.78 ± 0.20	100.00
Test item	60 min	1.74 ± 0.11	100.89
0.9% NaCl	60 min	1.72 ± 0.14	100.00

OD: Optical densitiy, mean of 6 values

SD: Standard deviation

Applicant's summary and conclusion

Interpretation of results:

other: Not corrosive

Conclusions:

The substance was tested for corrositivity in vitro according to OECD guideline 431 using reconstructed human skin as test system. The result revealed no corrosive property of the test item. There is regulatory acceptance in the EU that a substance can be considered corrosive (Skin Corrosive Cat.1A) based on a positive result in the human epidermis model test. Negative in vitro corrosivity responses are not conclusive with respect to non-classification or classification as irritant and shall therefore be subject to further evaluation.