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

Eye irritation

Administrative data

Endpoint:

eye 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 Guidelines no. 437: "Bovine corneal opacity and permeability (BCOP) test method for identifying ocular corrosives and severe irritants" (adopted September 07, 2009).

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 / tissue source

Species:

other: in-vitro test

Strain:

other: isolated bovine cornea

Details on test animals or tissues and environmental conditions:

TEST MATERIAL
- Source: slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands)
- Acclimation period: After receipt, the isolated corneas were stored at 32°C in a petri dish with cMEM (Eagle’s Minimum Essential Medium). The Bovine eyes were used as soon as possible after slaughter on the same day.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 32 +/- 1°C

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: Negative control: physiological saline; positive control: 20% (w/v) Imidazole solution prepared in physiological saline.

Amount / concentration applied:

An amount of 301 to 311 mg DFMMP was applied directly on the corneas in such a way that the cornea was completely covered.

Duration of treatment / exposure:

The corneas were incubated in a horizontal position for about 240 minutes at 32°C.

Observation period (in vivo):

Not applicable, the effects on the corneas was determined directly after the exposure period.

Number of animals or in vitro replicates:

Not applicable (in vitro study)

Details on study design:

The aim of this study was to evaluate the ocular irritancy of DFMMP as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea.

PROTOCOL
- The test was performed on a total of three corneas per treatment (DFMMP, negative and positive control).
- The opacity of each cornea was determined before exposure.
- The corneas were exposed for 240 minutes.
- After the incubation the change in opacity for each individual cornea (including the negative control) was determined.
- Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck) was evaluated.

INTERPRETATION
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the laboratory historical mean value.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the lab. historical range.
- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro irritancy score (IVIS)
- A test substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

Results and discussion

In vivo

Results

Irritation parameter:

other: The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score.

Remarks on result:

other: DFMMP did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 5.9 after 240 minutes of treatment.

Irritant / corrosive response data:

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 98 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
The in vitro irritancy scores ranged from 3.9 to 6.9 after 240 minutes of treatment with DFMMP. For this reason it can be concluded that DFMMP is not severe irritant or corrosive in the Bovine Corneal Opacity and Permeability test.