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Diss Factsheets

Toxicological information

Eye irritation

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Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
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 Type:
study report
Report date:

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
Eye irritation potential was determined using the in vitro SkinEthic Reconstituted Human Corneal model.

GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
3-bromo-1-(3-chloropyridin-2-yl)-1H-pyrazole-5-carboxylic acid
Constituent 2
Reference substance name:
Constituent 3
Reference substance name:
1H-Pyrazole-5-Carboxylic Acid, 3-Bromo-1-(3-Chloro-2-Pyridinyl)-
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, over silica gel

Test animals / tissue source

other: Human keratinocytes; cell line HCE (LSU Eye Center, New Orleans, USA)

Test system

unchanged (no vehicle)
other: Not applicable
Amount / concentration applied:
- Amount(s) applied (volume or weight with unit): 30 mg
Duration of treatment / exposure:
10 and 60 minutes
Observation period (in vivo):
Not applicable
Number of animals or in vitro replicates:
Not applicable
Details on study design:
-MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrolium bromide) Dulbecco's Phosphate Buffered Saline (DPBS) containing Ca++Mg++
-MTT Extractant (Isopropanol)
-Dulbecco's Phosphate Buffered Saline (DPBS) without Ca++Mg++: Used for tissue rinsing purposes.

-The test material was used as supplied.
-The negative control material, Solution A, was used as supplied. Solution A contained Glucose 1.802 g/L, Na2HPO4 0.142 g/L, HEPES 7.149 g/L, KCl 0.224 g/L and NaCl 7.597 g/L.
-The positive control material, SDS, was prepared as a 0.5% w/v solution in sterile distilled water.
-A 5.0 mg/mL MTT concentrate was prepared by adding 250 mg MTT to 50 mL of DPBS containing Ca++Mg++. The MTT concentrate was stored frozen. The concentrate was diluted to 0.5 mg/mL with SkinEthic maintenance medium when required.

The test material was checked for it's ability to reduce MTT directly. 25 mg of the test material was added to 1 mL of 0.5 mg/mL MTT solution freshly prepared in maintenance medium and incubated in the dark at room temperature for 60 minutes. Untreated MTT solution was used as a control. If the MTT solution color turned blue/purple relative to the control, the test material was presumed to have reduced the MTT.

Using sterile techniques, 800 uL of the maintenance medium at room temperature was dispensed into the appropriate number of wells of 6-well plates designated 'treatment plates'. Each well was labelled with details of the treatment and the appropriate exposure time. Separate treatment plates were used for each substance and negative and positive controls to avoid the possibility of cross contamination occurring. Before treatment, the Day 7 tissues were transferred from the 'arrival plates' into the wells of the 'treatment plates' containing the maintenance medium.

Triplicate tissues were treated with the test material for exposure periods of 10 minutes and 60 minutes. 30 mg of the test material was topically applied to the corresponding tissues. The tissues were dosed at regular timed intervals, to allow for the period taken to rinse each insert following exposure, and to ensure each tissue received an equal exposure time. Duplicate tissues were treated with 30 uL of solution A to serve as negative controls for the 10 and 60 minute exposure periods. Duplicate tissues were treated with 30 uL of SDS 0.5% w/v to serve as positive controls for the 10 and 60 minute exposure periods. In order to eliminate the possibility of volatile test materials affecting the viability of other treated tissues, the plates were sealed with a gas permeable sealing membrane. The plates were incubated at 37 degrees Celcius, 5% CO2 in air for the respective exposure times.

At the end of the relevant exposure period, each tissue insert was removed from the well using forceps and rinsed using a wash bottle containing DPBS. Rinsing was achieved by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper. Each tissue was placed into a pre-labelled 24-well plate designated 'holding plate' containing 300 µL of maintenance medium (at room temperature) until all the tissues were rinsed (including the negative and positive control treated tissues). Following rinsing of the tissues (two per group) they were transferred to a pre-labelled 24-well plate designated 'MTT Loading plate' containing 300 pl of a 0.5 mg/mL MTT solution freshly prepared in maintenance medium. The MTT loading plate was placed into an incubator for approximately three hours at 37 degrees Celcius, 5% CO2 in air.

At the end of the incubation period, the tissues were visually examined and the degree of MTT staining evaluated (qualitative evaluation of tissue viability). The inserts were blotted on absorbent paper to remove residual MTT solution and transferred to a pre-labelled 24-well plate designated 'MTT extraction plate' containing 0.75 mL of Isopropanol in each of a sufficient number of wells. An extra 0.75 mL of Isopropanol was added onto each tissue and the plate sealed to prevent Isopropanol evaporation. The plate was wrapped in aluminium foil (to protect from light) and allowed to stand overnight at room temperature to extract the formazan crystals out of the tissue.

At the end of the extraction period, each tissue insert was pierced with a pipette fitted with a 1000 uL tip and the extraction solution forced vigorously up and down through the tissue insert until a homogeneous solution was obtained. The empty inserts were discarded. For each tissue triplicate 200 uL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 uL of Isopropanol alone was added to three wells designated as 'blanks'. The optical density was measured (quantitative measurement of tissue viability) at 540nm (OD540) using the Anthos 2001 microplate reader.

One 10 minute test material treated tissue and one 60 minute test material treated tissue were retained for possible histology.

Results and discussion

In vivo

Irritant / corrosive response data:
The relative mean viability of the test material treated tissues was 88.1% after a 10 minute exposure and 72.2% after a 60 minute exposure.
It was considered unnecessary to proceed with tissue histology.

Following the 10 minute and 60 minute exposures the test material treated tissues appeared blue.
This was considered to be indicative of viable tissue.
Other effects:
Pre-test indicated that the test material was not able to directly reduce MTT.

Any other information on results incl. tables

A qualitative evaluation of tissue viability following the 10 minute and 60 minute exposures found that the test material treated tissues appeared blue. This was considered indicative of viable tissue.

Applicant's summary and conclusion

Interpretation of results:
other: Equivocal
Criteria used for interpretation of results: expert judgment
Using the SkinEthic Reconstituted Human Corneal model, the eye irritation potential as determined by an indicator of cytotoxicity based on reduction of MTT was found to be equivocal. The relative mean viability of the test material treated tissues was 88.1% after a 10 minute exposure and 72.2% after a 60 minute exposure.