2-chloropyridine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented, according to accepted guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD guideline 431 In vitro skin corrosion: human skin model test - reliability scoring based on 2004 guideline

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-chloropyridine

EC Number:

203-646-3

EC Name:

2-chloropyridine

Cas Number:

109-09-1

Molecular formula:

C5H4ClN

IUPAC Name:

2-chloropyridine

Details on test material:

- Name of test material (as cited in study report): 2-CHLOROPYRIDINE
- Physical state: Liquid (clear, colourless)
- Analytical purity: 99.2% (w/w)
- Lot/batch No.: 9RC123498
- Expiration date of the lot/batch: March 05, 2011
- Stability under test conditions: Not reported
- Storage condition of test material: Room temperature in the dark

Test animals

Species:

other: Adult human-derived epidermal keratinocytes (EPISKIN model kit)

Strain:

other: Not applicable

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Not applicable

ENVIRONMENTAL CONDITIONS
- Not applicable

Test system

Type of coverage:

other: None; test article was applied topically to tissues (in vitro)

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Controls:

other: positive and negative control groups were included

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL

Duration of treatment / exposure:

Single exposure; test groups were exposed for 3, 60, and 240 minutes
Single exposure; positive and negative control groups were exposed for 240 minutes

Observation period:

Not applicable. See details under study design.

Number of animals:

Not applicable.

Details on study design:

This test is based on the experience that corrosive chemicals are cytotoxic after a short term exposure to the EPISKIN model. Corrosive chemicals are able to penetrate the stratum corneum and are sufficiently cytotoxic to cause cell death in the underlying cell layers. Toxicity is determined by the metabolic conversion of the vital dye MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test article-treated cultures relative to the negative control.

Pre-test:
One limitation of the MTT assay is the possible interference of the test article with MTT (test article may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria). Therefore, to determine if the test article was able to directly reduce MTT, the test article was incubated with MTT solution freshly prepared in assay medium. Untreated MTT solution was used as a control.

The test article did not show any signs of direct MTT reduction; however, the results of the assay produced viabilities for the test article that were considerably greater than the concurrent negative control. Thus, a further step using water-killed tissues (which possess no metabolic activity but absorb and bind the test substance like viable tissues) was performed. The MTT-reducing test article was applied to a water-killed tissue for each exposure period and one water-killed tissue remained untreated. The untreated water-killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissue.

Main test:
Duplicate tissues were treated with the test article for exposure periods of 3, 60, and 240 minutes. Duplicate tissues were treated with the positive and negative control substances for an exposure period of 240 minutes. The test article (50 µL) was applied topically to the corresponding tissues ensuring uniform coverage of the tissues. Duplicate tissues were treated with 50 µL of 0.9% w/v sodium chloride solution, which served as negative controls. Duplicate tissues were treated with 50 µL of glacial acetic acid, which served as positive controls. At the end of each exposure period, each tissue was removed from the well and rinsed with dulbecco's phosphate buffered saline (PBS) with calcium and magnesium. The rinsed tissues were transferred into wells containing MTT solution. The tissues were incubated for 3 hours ± 5 minutes at room temperature (away from light). At the end of the 3-hour incubation period, each tissue was placed on absorbent paper to dry and the tissues were examined for the degree of MTT staining (qualitative evaluation of cell viability). Following qualitative evaluation of tissue viability, a total biopsy of the epidermis was taken using the EPISKIN biopsy punch. The epidermis was separated from the collagen matrix and both parts (epidermis and collagen matrix) was placed into micro tubes containing acidified isopropanol to extract formazan crystals out of the MTT-loaded tissues.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: Mean Optical Density of Duplicate Tissues

Value:

0.42

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 240 minutes. Reversibility: other: Not applicable for in vitro study. Remarks: Relative Mean Viability 206.4%; see details below. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: Mean Optical Density of Duplicate Tissues

Value:

0.43

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 60 minutes. Reversibility: other: Not applicable for in vitro test. Remarks: Relative Mean Viability 212.9%; see details below. (migrated information)

Results 3

Irritation / corrosion parameter:

other: other: Mean Optical Density of Duplicate Tissues

Value:

0.47

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 3 minutes. Reversibility: other: Not applicable for in vitro study. Remarks: Relative Mean Viability 232.7%; see details below. (migrated information)

In vivo

Irritant / corrosive response data:

Pre-test:
The test article was able to directly reduce MTT; therefore, an additional procedure using water-killed tissues was performed. The results obtained showed that no degree of interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.

Main study:
Following the 3, 60, and 240-minute exposure periods, the test article treated tissues appeared blue, which was considered by the authors to be indicative of viable tissue (based on results from the qualitative test).

Any other information on results incl. tables

The results are provided in Table 1.

Table 1: Mean Optical Density Values and Viabilities for the Negative Control, Positive Control, and Test Material
Material	Exposure Period (minutes)	Mean Optical Density of Duplicate Tissues	Relative Mean Viability (%)
Negative control	240	0.202	100*
Positive control	240	0.013	6.4
Test material	240	0.417	206.4
	60	0.430	212.9
	3	0.470	232.7

 *The mean viability of the negative control tissues is set at 100%.