Reaction product of {Sulfonic acid derivatives of [4-(aminoalkylamino)-6-substituted-1,3,5-triazin-2-ylamino]benzene}, ammonia water, sodium chloride and [poly(1~4)chlorosulfonyl(poly(1~4) benzo-poly(3~0)pyrido-5,10,15,20-tetraazaporphyrinato- N21,N22,N23,N24)]copper(II)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental starting date: 27 November 2013; Experimental completion date: 28 November 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1 as it has been conducted according to the Assessment of Ocular Irritation Potential using the SkinEthic Reconstructed Human Corneal Epithelial Model and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Assessment of Ocular Irritation Potential using the SkinEthic Reconstructed Human Corneal Epithelial Model (10-Minute Exposure)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification: CIM-25
Batch: MB-1
Expiry Date: 31 March 2014
Storage Conditions: room temperature in the dark

Test animals / tissue source

Species:

other: SkinEthic Reconstructed Human Corneal Epithelium Model

Strain:

other: Not applicable.

Details on test animals or tissues and environmental conditions:

Not applicable for test animals:

Human Corneal Epithelium (HCE) model supplied by SkinEthic Laboratories, Lyon, France

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: Not applicable.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Tissues were treated with 30 mg of the test item.

Duration of treatment / exposure:

10 minutes.

Observation period (in vivo):

Not applicable.

Number of animals or in vitro replicates:

Not applicable.

Details on study design:

PREPARATION OF NEGATIVE AND POSITIVE CONTROL ITEMS:
The negative control item, Solution A (defined below), was used as supplied.
The positive control itme, Sodium Dodecyl Sulphate 2% w/v, was prepared in sterile water.

Solution A Composition for 1 Liter
Na2HPO4: 0.142 g/L
Glucose: 1.802 g/L
HEPES: 7.149 g/L
KCl: 0.224 g/L
NaCl: 7.597 g/L

PRE-TEST - ASSESSMENT OF DIRECT TEST ITEM REDUCTION OF MTT:
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.

One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT to formazan, thus mimicking dehydrogenase activity of the cellular mitochondria of viable cells. This property of the test item 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 item on or in the tissues. To identify this possible interference, the test item was checked for its ability to reduce MTT directly.

30 mg of test item was added to 1 mL of a 0.5 mg/mL MTT solution and incubated at 37°C, 5 % CO2 in air for 3 hours. Untreated MTT solution was used as a control. If the MTT solution turned blue, the test item was presumed to have reduced the MTT.

The test item was found to have the ability to directly reduce MTT and therefore the following procedure, employing freeze-killed tissues that possess no metabolic activity but absorb and bind the test item like viable tissues, was required.

In addition to the normal test procedure, the MTT reducing test item was applied to one freeze killed tissue. One freeze-killed tissue remained untreated. The untreated freeze-killed tissue demonstrates a small amount of MTT reduction due to residual reducing enzymes.

Freeze-killed tissues were prepared by placing untreated SkinEthic HCE tissues in a freezer (-14 to -30 ºC) overnight. Once killed, the tissues were stored in the freezer.

RECEIPT AND PREPARATION 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.

MAIN TEST:
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 labeled with details of the treatment and the appropriate exposure time. Separate treatment plates were used for the test item, 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.

Triplicate tissues were treated with 30 mg of the test item 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 2 % 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) without Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper. Each tissue was placed into a pre-labeled 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 were transferred to a pre-labeled 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.

Due to the intrinsic 'blue' color of the test item a further limitation of the assay is possible color interference with measurement of the extracted MTT. Therefore in addition to the application procedure described above, two additional test item treated tissues and one additional color correction negative control tissue (Solution A) were treated to evaluate possible interference. These tissues followed the same treatment steps as the viable tissues except for the MTT step where MTT incubation is replaced by incubation with fresh maintenance medium for the color correction tissues.

At the end of the incubation period the inserts were rinsed twice with phosphate buffered saline and blotted on absorbent paper to remove residual MTT solution and transferred to a pre-labeled 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 aluminum 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-labeled 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 562nm (OD562) using the Anthos 2001 microplate reader.

Results and discussion

In vivo

Results

Irritation parameter:

other: Relative Mean Viability (%)

Basis:

mean

Remarks:

(Test item)

Time point:

other: 10 minutes

Score:

99.7

Remarks on result:

other: Non-irritant.

Irritant / corrosive response data:

Assessment of Eye Irritation Potential:
The individual and mean OD562 values and mean viabilities for each treatment group are given in Table 1.

The relative mean viability of the test item treated tissues after a 10 Minute exposure period was 99.7%.

Other effects:

Assessment of Direct Test Item Reduction of MTT:
The MTT solution containing the test item turned purple which indicated that the test item directly reduced MTT and therefore the MTT viability assay was performed in parallel on viable and freeze-killed tissues. However, the results obtained showed a negligible degree of interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results or for reporting purposes.

Color Interference Check:
It was decided that color correction tissues should be incorporated into the testing due to the intrinsic 'dark blue' color of the test item. The color correction tissues would be used to measure any staining, adherence or absorbed test item into the tissue that may have caused interference with the optical density measurements.

Therefore in addition to the normal dosing procedure, an additional two tissues were treated with the test item. A further tissue was treated with the negative control item, solution A, for correcting background color. These additional groups were not placed into MTT but into maintenance medium for the equivalent MTT exposure of 3 hours.

The optical density results indicated that negligible color interference had occurred due to the colored test item and therefore the additional tissues were not required for correction of the results.

Any other information on results incl. tables

Table 1: Assessment of Eye Irritation Potential - Viability of HCE Tissues

Item	OD562 of Individual Tissue	Mean OD562	Relative Mean Viability (%)
Negative Control	1.075	1.098	100*
	1.085
	1.135
Positive Control	0.177	0.146	13.3
	0.127
	0.135
Test Item	1.108	1.095	99.7
	1.098
	1.080

* = 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: EU

Conclusions:

According to the study plan followed the test item was considered to be Non-Irritant.
ED DSD (67/548/EEC): Not classified for irritation.
ED CLP (1272/2008/EC)/UN GHS: Not classified for irritation.

Executive summary:

Introduction

The purpose of this study was to determine the eye irritation potential of the test item using the SkinEthic reconstructed Human Corneal Epithelium model (HCE, SkinEthic Laboratories, Lyon, 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.

Method

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 item followed by the main test.

For the main test, triplicate SkinEthic tissues were treated with the test item for 10 minutes. At the end of the exposure period each SkinEthic tissue was rinsed. The rinsed tissues were taken for MTT loading. After MTT-loading the tissues were removed and immersed in isopropanol for extraction of formazan crystals out of the MTT-loaded 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 562 nm (OD₅₆₂). Data are presented in the form of percentage viability (MTT conversion relative to negative controls).

The mean tissue viability for the test item was compared to the negative control and classified according to the following table:

Criteria for in vitro interpretation	Prediction	EU DSD (67/548/EEC)	EU CLP (1272/2008/EC)	UN GHS
Relative mean tissue viability≤60%	Irritant	Symbol “Xi” Risk Phrase R36 or R41	H318 or H319 Category 1 or 2	H318, H319 or H320 Category 1or 2A or 2B
Relative mean tissue viability >60%	Non-Irritant	Not classified for irritation	Not classified for irritation	Not classified for irritation

Results

The relative mean viability of the test item treated tissues after a 10 Minute exposure period was 99.7%.

Quality Criterion

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

Conclusion

According to the study plan followed the test item was considered to be Non-Irritant.

ED DSD (67/548/EEC): Not classified for irritation.

ED CLP (1272/2008/EC)/UN GHS: Not classified for irritation.