2,3,5,6-tetrachloropyridine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 July 2009 to 16 July 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The purpose of this study was to determine the eye irritation potential of the test material using the SkinEthic Reconstituted Human Corneal model (HCE, SkinEthic Laboratories, Nice, France) after a treatment period of 10 minutes (1, 2).
The SkinEthic RHC model consists of transformed human keratinocytes of the cell line HCE (LSU EYE Center, New Orleans, USA) that form a corneal epithelial tissue (mucosa), devoid of stratum corneum, resembling, histologically, the mucosa of the human eye. Test materials are applied directly to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. The model consists of an airlifted, living, corneal tissue construct, produced in polycarbonate inserts in serum-free and chemically defined medium.
The test is based on the hypothesis that irritant chemicals are able to penetrate the stratum corneum of the SkinEthic RHC model and are sufficiently cytotoxic to cause cell death in the underlying cell layers.
Cytotoxicity was determined by the reduction of MTT to formazan by viable cells in the test material treated tissues (quantitative measurement of tissue viability) relative to the negative control.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,3,5,6-tetrachloropyridine

EC Number:

219-283-9

EC Name:

2,3,5,6-tetrachloropyridine

Cas Number:

2402-79-1

Molecular formula:

C5HCl4N

IUPAC Name:

2,3,5,6-tetrachloropyridine

Details on test material:

Batch number XF09160201 of purity = 99.4%

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

Not applicable

Test system

Amount / concentration applied:

Not applicable

Duration of treatment / exposure:

Not applicable

Observation period (in vivo):

Not applicable

Number of animals or in vitro replicates:

Not applicable

Details on study design:

Pre-Test

Assessment of Direct Test Material Reduction of MTT

One limitation of the assay is possible interference of the test material with MTT. A test material may directly reduce MTT to formazan, thus mimicking dehydrogenase activity of the cellular mitochondria of viable cells. This property of the test substance is only a problem, if at the time of the MTT test (after the chemical has been rinsed off) there are still sufficient amounts of the test material on or in the tissues. To identify this possible interference, the test material was checked for its ability to reduce MTT directly.
30 mg of test material was added to a 0.5 mg/ml MTT solution and incubated at room temperature in the dark for 60 minutes. Untreated MTT solution was used as a control. If the MTT solution turned black/purple, the test material was presumed to have reduced the MTT.

Receipt of Tissues

On arrival, the SkinEthic HCE tissues (Day 6 cultures), were stored at room temperature prior to transferring into 24-well plates designated ‘arrival plates’ containing 300 µl of maintenance medium. It was important to ensure that there were no air bubbles present under the tissue inserts. The tissues were incubated overnight at 37°C, 5% CO2 in air.

Preparation of Tissues

Using sterile techniques, 1 ml of 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 the test material and negative and positive controls to avoid the possibility of cross contamination occurring. Before treatment, the 7 day old tissues were transferred from the ‘arrival plates’ into the wells of the ‘treatment plates’ containing the maintenance medium.

Main Test

Triplicate tissues were treated with 30 mg of the test material for 10 minutes. The tissues were dosed at regular timed intervals to allow for the periodtaken to rinse each insert following exposure and to ensure each tissue received an equal exposure time. Triplicate tissues were treated with 30 µl of olution A to serve as negative controls and triplicate tissues were treated with 30 µl of 1 % w/v SDS to serve as positive controls. The plates were incubated at 37°C, 5% CO2 in air during the exposure time.
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 Dulbecco’s Phosphate Buffered Saline (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. Following rinsing, the tissues (two per group) were transferred to a pre labelled 24 well plate designated ‘MTT Loading plate’ containing 300 µl 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°C, 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 µl 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 µl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µl 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.

Tissue Histology

One tissue for each treatment group was retained for possible tissue histopathology.
The tissues were carefully cut out of the polycarbonate inserts with a sharp scalpel. The tissues were carefully cut in half. Both halves were placed into a pre-labelled 1.5 ml Eppendorf tube containing 1 ml of 10% Formalin and stored at room temperature.

Results and discussion

In vivo

Results

Irritation parameter:

other: Reduction of MTT

Time point:

other: 10 minutes

Score:

128.8

Remarks on result:

other: The score given is the relative mean viability of the test material treated tissues in % terms.

Irritant / corrosive response data:

Assessment of Direct Test Material Reduction of MTT

The test material was not able to directly reduce MTT.

Assessment of Eye Irritation Potential

The mean OD540 values and mean viabilities for each treatment group are given in Table 1.
The relative mean viability of the test material treated tissues after a 10 minute exposure was 128.8%.
It was considered unnecessary to proceed with tissue histopathology.

Qualitative Evaluation of Tissue Viability (MTT Uptake Visual Assessment)

The qualitative evaluation of tissue viability is presented in Table 2.
The test material and negative control material treated tissues appeared blue which was considered to be indicative of viable tissue. The positive control material treated tissues appeared blue/white which was considered to be indicative of semi viable tissue.

Assay Acceptance Criterion

The quality criterion required for the acceptance of results in the test was satisfied.

CONCLUSION

According to the protocol followed the test material was considered to be a Non-Irritant (NI).

Any other information on results incl. tables

Table1              Assessment of Eye Irritation Potential – Viability of RHC Tissues

Material	Mean Tissue Viability	Mean OD540	Viability (%)
Negative Control	0.977	0.961	100*
	0.944
Positive Control	0.704	0.652	67.8
	0.599
Test Material	1.164	1.238	128.8
	1.312

 

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

Table2              Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material	Score
	Tissue 1	Tissue 2
Negative Control	-	-
Positive Control	+	+
Test Material	-	-

MTT Visual Scoring Scheme of SkinEthic Tissues

-     =  Blue tissue (viable)

+    =  Blue/White tissue (semi viable)

++  =  Tissue completely white (dead)

 

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

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: In accordance with the principles of the test method

Conclusions:

According to the protocol followed the test material was considered to be a Non-Irritant.

Executive summary:

The eye irritation potential of the test material was evaluated using the SkinEthic Reconstituted Human Corneal model (HCE, SkinEthic Laboratories) after a treatment period of 10 minutes. The substance was considered to be Non-Irritant under the conditions of the test.