Copper phtalocyanine monosulfonated and disulfonated

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18.9 - 8. 10. 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was performed according to recommended method in accordnace with GLP principles

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Principles of method if other than guideline:

In vitro test on bovine cornea.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Copper phtalocyanine monosulfonated and disulfonated

EC Number:

700-870-8

Molecular formula:

C32H15N8Cu(HSO3)n where n=1.3-1.6

IUPAC Name:

Copper phtalocyanine monosulfonated and disulfonated

Constituent 2

Reference substance name:

Copper phtalocyanine mono- and disulphonated

IUPAC Name:

Copper phtalocyanine mono- and disulphonated

Constituent 3

Reference substance name:

Hysperse 12

IUPAC Name:

Hysperse 12

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Hysperse 12
Copper phtalocynanine mono- and disulphonated
- Molecular formula: C32H15N8Cu(SO3H)1.4x2.5H20
- Molecular weight:733.3
- Substance type:multicomponpowderviscous liquid
- Lot/batch No.: 01
- Stability under test conditions: stable
- Storage condition of test material: The substance was stored in closed vessel in dry room at the temperature bellow 25ºC in dark.

Test animals / tissue source

Species:

other:

Details on test animals or tissues and environmental conditions:

No animals, in vitro test on bovine cornea
Corneas free of defects were dissected with a 2 to 3 mm rim of sclera remaining to assist in subsequent handling, with care taken to avoid damage to the corneal epithelium an endothelium. Isolated corneas were mounted in specially designed corneal holders that consisted of anterior and posterior compartments, which interfaced with the epithelial and endothelial sides of the cornea, respectively.

Test system

Vehicle:

water

Amount / concentration applied:

The test substance was tested as solution at 20% concentration in a 0.9% sodium chloride solution. 2g of the test substance was dissolved in 10mL of 0.9% sodium chloride.
750 μL was used to cover the epithelial side of the cornea.

Duration of treatment / exposure:

see details on study design

Details on study design:

Procedure scheme:
Selection of corneas, mounting in holders → incubation with EMEM (Eagle`s Minimum Essential Medium 1hour (32 ± 1°C) → removed EMEM, measurement of baseline opacity → treatment by positive and negative control substance and test substance (incubation 10 min.) → washing epithelium, incubation 2 hour (32 ± 1°C), measurement of opacity after application → application of sodium fluorescein (4 mg/ml), incubation 1.5 hour (32 ± 1°C) → measurement of absorbance (490 nm).

Preparation of the eyes:
Corneas free of defects were dissected with a 2 to 3 mm rim of sclera remaining to assist in subsequent handling, with care taken to avoid damage to the corneal epithelium an endothelium. Isolated corneas were mounted in specially designed corneal holders that consisted of anterior and posterior compartments, which interfaced with the epithelial and endothelial sides of the cornea, respectively. Both chambers were filled to excess with
pre-warmed Eagle's Minimum Essential Medium (EMEM). The device was then equilibrated at 32 ± 1°C for at least one hour in water bath to allow the corneas to equilibrate with the medium and to achieve normal metabolic activity, to the extent possible. Following the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Any corneas that showed macroscopic tissue
damage (e.g., scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded. The mean opacity of all equilibrated corneas was calculated.

Treatment groups:
The test was performed using 9 isolated bovine corneas. 3 corneas for each group – positive control group, negative control group and test substance group.

Application form preparation:
The test substance was tested as solution at 20% concentration in a 0.9% sodium chloride solution. 2g of the test substance was dissolved in 10mL of 0.9% sodium chloride.

Control substances
Concurrent negative controls and positive controls were included in experiment. The control group was included in the BCOP test method so that nonspecific changes in the test system could be detected and to provide a baseline for the assay endpoints
Negative control substance: 0.9% sodium chloride solution
Positive control substance: 20 % imidazole

Aplication of the substance:
Closed-chamber method was used, because the test substance was applicable by micropipette. The test substance (750 μL) to cover the epithelial side of the cornea is introduced into the anterior chamber through the dosing holes on the top surface of the chamber, and the holes were subsequently sealed with the chamber plugs during the exposure.

Post-exposure:
After the exposure period, the test substance, the negative control, or the positive control substance was removed from the anterior chamber with EMEM (containing phenol red - the effectiveness of rinsing acidic or alkaline materials). The corneas were given a final rinse
with EMEM (without phenol red). The EMEM (without phenol red) was used as a final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded.

Endpoints Measured:
Opacity - the amount of light transmission through the cornea. Corneal opacity was measured quantitatively with the aid of an opacitometer (Opacitometer, MC2 - Le spécialiste du laboratoire – France) resulting in opacity values measured on a continuous scale.
Permeability - the amount of sodium fluorescein dye that penetrates all corneal cell layers (i.e., the epithelium on the outer cornea surface through the endothelium on the inner cornea surface) measured indirectly using UV/VIS visible light spectrophotometry (at 490 nm).
1 mL sodium fluorescein solution (5 mg/mL) was added to the anterior chamber of the corneal holder, which interfaced with the epithelial side of the cornea, while the posterior chamber, which interfaced with the endothelial side of the cornea, is filled with fresh EMEM. The holder was incubated in
horizontal position for 1.5 hours at 32 ± 1 ºC
The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured with the aid of UV/VIS spectrophotometry (Spektrofotometer GENESYSTM 10 UV/VIS Scanning). The values of absorbance measured at 490 nm were recorded as optical density (OD490) values. This term was used because the measuring is performed with visible light spectrophotometer using a standard 1 cm path length.

Evaluation of results
Mean opacity:
Opacity values of treated corneas were corrected by subtracting individual background opacity values and the mean opacity is calculated.
Mean permeability:
Mean OD value of treated corneas was corrected by subtracting the mean OD value of negative control and the mean opacity is calculated.
IVIS calculation:
Resulting mean opacity and OD490 values for each treatment group was combined in an empirically-derived formula to calculate an in vitro irritancy score (IVIS) for each treatment group as follows:
IVIS = mean opacity value + (15 x mean permeability OD490 value)

Decision criteria:
A substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

Results and discussion

In vivo

Results

Irritation parameter:

other: IVIS (In Vitro Irritancy Score ) - see section Details on study design

Basis:

other: see section Details on study design

Time point:

other: 1.5 h

Score:

30.06

Reversibility:

not specified

Irritant / corrosive response data:

Test substance was not identified as corrosive or severe irritant.

Any other information on results incl. tables

4. 2. Appearance of corneas after the test substance exposure
Table 2: Appearance of corneas

 

Group	Cornea No.	Appearance after exposure
Test substance	1	Without macroscopic damage, mild colouring of the test substance
	6	Without macroscopic damage, mild colouring of the test substance
	14	Without macroscopic damage, mild colouring of the test substance
Negative control	3	Without macroscopic damage
	10	Without macroscopic damage
	12	Without macroscopic damage
Positive control	2	Corneal opacity
	4	Corneal opacity
	9	Corneal opacity

 

Appearance of corneas was observed before and after application of the test substance,negative and positive control

(see table 1 and 2).No macroscopic damage was observed on corneas before application.Corneal opacity was observed

on the corneas treated by positive control. The corneas treatedby negative control were without macroscopic damage.

The corneas treated by the testsubstance were without macroscopic damage but corneas were mild coloured by the testsubstance.

4.3. Opacity

The opacity values were measured after initial incubation with EMEM (baseline opacity) and after treatment by substances.

Table 3: Opacity values

Group	Cornea No.	Baseline opacity	Opacity after treatment	Opacity difference	Mean opacity difference
NC(0.9% NaCl)	3	6	7	1	0.33
	10	5	5	0
	12	5	5	0
PC(20% Imidazole)	2	4	57	53	48.67
	4	4	54	50
	9	6	49	43
EXP(Hysperse 12)	1	4	36	32	30.00
	6	6	34	28
	14	5	35	30

 

Note:NC-negative control, PC-positive control, EXP – test substance application form

4.4. Permeability

The amount of sodium fluorescein that crosses into the posterior chamber was measured with the aid of UV/VIS spectrophotometry

(see Tab. 3).

Table 4: Optical density values

Group	Cornea No.	Optical density (490nm)	Mean optical density
NC(0.9% NaCl)	3	0.013	0.009
	10	0.012
	12	0.002
PC(20% Imidazole)	2	1.682	1.770
	4	1.853
	9	1.776
EXP(Hysperse 12)	1	0.025	0.013
	6	0.003
	14	0.012

 

Note:NC-negative control, PC-positive control, EXP – test substance application form

4.5. Evaluation of results

Data treatment:

The In Vitro Irritancy Score(IVIS) was computed according the following formula: IVIS = mean opacity value +

(15 x mean permeability OD490 value)

Table 5: IVIS values

Group	IVIS
	Calculation	Result
NC(0.9% NaCl)	0.33 + 15 x 0.009	0.47
PC(20% Imidazole)	48.67 + 15 x (1.770 -0.009)	75.09
EXP(Hysperse 12)	30.00 + 15 x (0.013 – 0.009)	30.06

 

Note:NC-negative control, PC-positive control, EXP – test substance application form

After exposure the washing of corneas is performed. In spite of this procedure the corneas treated by the test substance

remain mildly coloured. This colouring could influence the measuring of opacity what could result in higher opacity

values. Nevertheless the final IVIS was negative, so no corrections should be made.

Decision criteria:

Limit value: IVIS≥55.1=positive response

The result of study:IVIS = 30.06

Výzkumný ústav organických syntéz a.s., CETA Hysperse 12 – BCOP
Study acceptance criteria:
Table 6: IVIS – control historical value for positive control

 

Control	Mean	Standard deviation	Upper limit	Lower limit
20% Imidazole	76.94	7.22	84.17	69.72

 

Notes:Upper limit = mean+one standard deviations of the current historical mean Lower limit=mean-one standard

deviations of the current historical mean The historical means of IVIS are updated at least every three months.

The value of IVIS for positive control (20% Imidazole) obtained during the study was 75.09. This value is within the

acceptance limit (one standard deviations of the current historical mean), sothe study is considered acceptable.

Table 7: Opacity and Permeability -Control historical values for negative control

Value	Control	Mean	deviation Standard	Upper limit
Opacity	0.9% NaCl	1.07	1.03	2.11
Permeability	0.9% NaCl	0.0301	0.0439	0.0740

 

Notes: Upper limit = mean+one standard deviation of the current historical mean The historical means of opacity

and permeability are updated at least every three months.

The value of opacity for negative control (0.9% NaCl) obtained during the study was 0.33and value of permeability

was 0.009.The values obtained during this study not exceeded upper limits, so thestudy is considered acceptable.

Applicant's summary and conclusion

Interpretation of results:

other: The substance is not corrosive or severe irritant

Remarks:

Criteria used for interpretation of results: EU

Executive summary:

The test substance, Hysperse 12, was tested for the evaluation the potential ocular corrosivity yor severe irritancy as measured by its ability to induce opacity and increased permeability in an isolated bovine cornea. The test was performed according to the Method B.47 Bovine Corneal Opacity and dPermeability Test Method for Identifying Ocular Corrosives and Severe Irritants, Council

lRegulation (EC) No.1152/2010.

The test was performed using nine isolated bovine corneas. The testing was performed on three groups of corneas: test substance treatment group, positive control group and negative control group. Three corneas per group were used.

Closed-chamber method was used, because the test substance was applicable by micropipette. The opacity and permeability of each cornea were measured. The In Vitro Irritancy Score e(IVIS) was calculated from the values of opacity and permeability.

The In Vitro Irritancy Score (IVIS) for Hysperse 12 was 30.06. This is less than the limit value of IVIS (55.1), which means that the test substance was not identified as a corrosive or severe irritant.