1H-Pyrazole-5-carboxylic acid, 3-bromo-1-(3-chloro-2-pyridinyl)-

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Not a standard, validated method but is well documented and scientifically valid; GLP study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Skin irritation potential was evaluated using the EPISKIN reconstituted human epidermis model.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

481-150-8

EC Name:

-

Cas Number:

500011-86-9

Molecular formula:

C9H5BrClN3O2

IUPAC Name:

3-bromo-1-(3-chloropyridin-2-yl)-1H-pyrazole-5-carboxylic acid

Constituent 2

Reference substance name:

DBC80

IUPAC Name:

DBC80

Constituent 3

Reference substance name:

1H-Pyrazole-5-Carboxylic Acid, 3-Bromo-1-(3-Chloro-2-Pyridinyl)-

IUPAC Name:

1H-Pyrazole-5-Carboxylic Acid, 3-Bromo-1-(3-Chloro-2-Pyridinyl)-

Details on test material:

- Name of test material (as cited in study report): IN-DBC80
- Physical state: off white solid
- Lot/batch No.: CHCAEDOOOl
- Storage condition of test material: room temperature in the dark, under silica

Test animals

Species:

other: human-derived epidermal keratinocytes

Details on test animals or test system and environmental conditions:

Not applicable

Test system

Type of coverage:

other: in vitro

Preparation of test site:

other: in vitro

Vehicle:

unchanged (no vehicle)

Controls:

other: Not applicable

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 +/- 2 mg

Duration of treatment / exposure:

15 +/- 0.5 minutes

Observation period:

42 hours

Number of animals:

Not applicable

Details on study design:

PREPARATION OF TEST MATERIAL
The test material was used as supplied.

POSITIVE CONTROL
Sodium Dodecyl Sulphate (SDS)

NEGATIVE CONTROL
Dulbecco's Phosphate Buffered Saline (PBS) with Ca++ and Mg++

ASSESSMENT OF DIRECT TEST MATERIAL REDUCTION OF MTT
- MTT dye metabolism, cell viability assay
The MTT assay, a colourimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazoliumbromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells in the test material treated tissues relative to the negative controls.

-Assessment of Direct Test Material Reduction of MTT
One limitation of the assay is possible interference of the test substance with MTT. A test substance may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test substance is only a problem if at the time of the MTT test (after rinsing), there is still sufficient amounts of the test substance present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by the procedure described below.

- Test for Direct MTT Reduction
As specified, a test substance may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, each test substance is checked for the ability to directly reduce MTT according to the procedure below:
Step 1: 10 mg of the test material was added to 2 mL of a 0.3 mg/mL MTT solution (v/v) freshly prepared in assay medium. The solution was incubated in the dark at 37 degrees Celcius, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control. If the MTT solution containing the test substance turns blue/purple, the test substance is presumed to have reduced the MTT.

-Pre-Incubation (Day 0: tissue arrival)
2 mL of maintenance medium, warmed to approximately 37 degrees Celcius, was pipetted into the first column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for each test material and control material. The tissues were incubated at 37 degrees Celcius, 5% CO2 in air for at least 24 hours.

MAIN TEST
- Application of Test Material and Rinsing (Day 1)
2 mL of maintenance medium, warmed to approximately 37 degrees Celcius, was pipetted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with the test material for an exposure period of 15 minutes. The test material was applied topically to the corresponding tissues to ensure uniformity in covering of the tissues. 5 uL of sterile distilled water was topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. 10 +/- 2 mg of the solid test material was then applied. Triplicate tissues, treated with 10 uL of PBS, were used to serve as negative controls. Triplicate tissues, treated with 10 uL of SDS 5% w/v, were used to serve as positive controls. To ensure satisfactory contact with the positive control material, the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After 7 minutes contact time, the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period. The plate(s) were kept in the biological safety cabinet at room temperature for 15 + 0.5 minutes.

At the end of the exposure period, each tissue was removed from the well using forceps and rinsed with 25 mL of PBS. Rinsing was achieved by filling and emptying each tissue insert with PBS using a pipette. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 degrees Celcius, 5% C02 in air for approximately 42 hours.

-MTT Loading/Formazan Extraction (Day 3)
Following the 42-hour post-exposure incubation period, each 12-well plate was placed onto a plate shaker for 15 +/- 2 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30°Celcius for possible inflammatory mediator determination. 2 mL of 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12 well plate(s). The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 degrees Celcius, 5% C02 in air. At the end of the 3 hour incubation period, each tissue was placed onto absorbent paper to dry. The tissues were examined and the degree of MTT staining evaluated (qualitative evaluation of cell viability) using the MTT Visual Scoring scheme. Following qualitative evaluation of tissue viability, a total biopsy of the epidermis was made using the EPISKIN biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10°Celcius until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

- Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution. For each tissue, duplicate 200 uL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 uL of acidified isopropanol alone was added to the two wells designated as 'blanks'. The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

DETERMINATION OF IL-1 alpha CONCENTRATION IN CULTURE MEDIUM
In response to physical or chemical stress, keratinocytes produce and release a number of cytokines which rapidly generate cutaneous inflammation.This observation allows for the measurement of such keratinocyte responses to evaluate toxicological properties of chemicals in order to identify irritants. A complimentary endpoint, IL-1 alpha release is incorporated in this assay for those chemicals which do not reduce tissue viability below the threshold for predicting irritancy (50%). This complimentary endpoint was used to either confirm a non-irritant result or used to override the non-irritant result thereby improving the sensitivity of the assay.

-Procedure: For test materials showing a relative tissue viability of >50%, the amount of IL-1 alpha (pg/mL) released into the culture medium at the end of the 42-hour post-exposure incubation period was measured in duplicate using R&D systems IL-1 alpha ELISA kit DLA50 (R&D systems, Abingdon, UK). A detailed test procedure was supplied with the kit.

-Interpretation of Result: The test material is considered to be an irritant if the relative tissue viability after 15 minutes exposure and 42 hours post-exposure incubation is > 50% and the amount of IL-1 alpha release is > 60 pg/mL. The test material is considered to be a non-irritant if the relative tissue viability after 15 minutes of exposure and 42 hours of post-exposure incubation is > 50% and the amount of IL-1 alpha released is less than or equal to 60 pg/mL.

Results and discussion

In vivo

Irritant / corrosive response data:

The relative mean viability of the test material treated tissues was 109.7% after a 15-minute exposure.
Following the 15-minute exposure the test material treated tissues appeared blue which was considered indicative of viable tissue.

Other effects:

DIRECT MTT REDUCTION
The MTT solution containing the test material did not turn blue/purple which indicated that the test material did not directly reduce MTT.

Any other information on results incl. tables

The concentration of inflammatory mediator IL-1 alpha in the culture medium retained from the test material treated tissues was 20.485 pg/mL. This result confirmed the absence of cytotoxicity as determined in the MTT assay.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: EPISKIN Reconstituted human epidermis model

Conclusions:

Using the EPISKIN reconstituted human epidermis model, the skin irritation potential of the test material as determined by an indicator of cytotoxicity based on reduction of MTT was found to be non-irritating. This result was confirmed by assessment of the inflammatory mediator IL- 1 alpha.