2,4-Thiophenedicarbonitrile, 5-amino-3-methyl-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 January 2010 to 21 January 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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.
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. The test material is 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.
One tissue for each treatment group was retained for possible tissue histopathology.

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 period taken to rinse each insert following exposure and to ensure each tissue received an equal exposure time. Triplicate tissues were treated with 30 µl of solution 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.

* Solution A Composition for 1 Litre
- Na2HPO4 0.142 g/I
- Glucose 1.802 g/I
- HEPES 7.149 g/I
- KCI 0.224 g/I
- NaCl 7.597 g/l

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

5-amino-3-methylthiophene-2,4-dicarbonitrile

EC Number:

610-868-8

Cas Number:

52603-48-2

Molecular formula:

C7N3H5S

IUPAC Name:

5-amino-3-methylthiophene-2,4-dicarbonitrile

Test animals / tissue source

Species:

other: Reconstituted Human Corneal model

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

Not applicable. Test was conducted in vitro.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

30 mg

Duration of treatment / exposure:

10 minutes

Observation period (in vivo):

Not applicable

Number of animals or in vitro replicates:

No animals
Test was carried out in vitro.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done):
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.
- Time after start of exposure:
10 mins.

SCORING SYSTEM:
Optical density

TOOL USED TO ASSESS SCORE:
Anthos 2001 microplate reader.

Results and discussion

In vivo

Results

Irritation parameter:

other: mean % viability

Basis:

mean

Time point:

other: 10 minutes

Score:

101.7

Reversibility:

other: not applicable

Remarks on result:

other: In vitro result using the SKINETHIC reconstituted human corneal epithelium model.

Irritant / corrosive response data:

RESULTS
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 below.
The relative mean viability of the test material treated tissues after a 10 minute exposure was 101.7%.
It was considered unnecessary to proceed with tissue histopathology.

Other effects:

No information provided.

Any other information on results incl. tables

Assessment of Eye Irritation Potential – Viability of RHC Tissues

Material	Mean Tissue Viability	Mean OD540	Viability (%)
Negative Control	0.894	0.920	100*
	0.945
Positive Control	0.352	0.384	41.7
	0.415
Test Material	0.918	0.936	101.7
	0.954

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

 

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)

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: not able to directly reduce MTT and relative mean tissue viability was >60

Conclusions:

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

Executive summary:

Introduction

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. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death.

 

Methods

The experimental design of the study consists of a test for direct reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) by the test material followed by the main test.

For the main test, triplicate SkinEthic tissues were treated with 30 mg of the test material for 10 minutes. Triplicate tissues treated with 30 µl of Solution A served as the negative control and triplicate tissues treated with 30 µl of 1% w/v Sodium Dodecyl Sulphate served as the positive control.

At the end of the exposure period each SkinEthic tissue was rinsed. The rinsed tissues (two per group) were taken for MTT loading. The remaining tissues were retained for possible histopathology. Following MTT loading the reduced MTT was extracted from the tissues.

After extraction the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured. The optical density was measured at 540 nm (OD₅₄₀). Data are presented in the form of percentage viability (MTT conversion relative to negative controls).

The test material was classified according to the following criteria:

i)                   If the percentage relative mean tissue viability was ≥60% the test material was considered to be non-irritant.

ii)                 If the percentage relative mean tissue viability was <60% the test material was considered to be an irritant.

 

Results

The relative mean viability of the test material treated tissues after a 10 minute exposure was 101.7%.

It was considered unnecessary to proceed with tissue histopathology.

 

Quality criteria

The quality criteria required for acceptance of results in the test were satisfied.

 

Conclusion

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