Copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, sulfonated

Endpoint summary

Description of key information

Skin irritation:

(1) In a recent acute dermal toxicity study, no significant irritation was reported at the limit concentration of 2,000 mg/kg bw. Only very slight edema was noted for 1 male on Study Day 3 and very slight and/or slight erythema was noted for all females during Study Days 0 - 4; animals did not show signs of irritation for the remainder of the study.

(2) The test substance was assayed for the in vitro skin corrosion in human epidermal model EpiDermTM, according to Method B.40. Skin corrosion (in vitro),Council Regulation (EC) No.440/2008. The test substance (25 mg) was placed atop the previously moistened tissue, with nine tissues for the experiment (i.e. three per test substance (C1), three for positive control (PC) and three for negative control (NC)) and exposure times of 3 and 60 minutes. After rinsing, tissues were incubated with MTT for three hours and extracted overnight subsequently at room temperature without shaking. OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues. As the test substance was suspected to be direct reducing, the test with frozen tissues was performed to detect if the test substance actually reduces MTT in tissues directly. Because this test excluded initial suspicion, the correction of MTT test results has not been made. Based on the experimental design, average viability of tissues treated by the test substance was 109.5% of negative control average value after 3 min treatment and 76.5 % after 60 min treatment. The test results concluded the test substance was non-corrosive in EpiDermTM model.

(3) The test substance was assayed for the in vitro skin irritation in human epidermal model EpiDermTM, according to Method B.46. In vitro skin irritation: Reconstructed human epidermis model test and Protocol for: In Vitro EpiDermTM Skin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT. After pre-incubation of tissues, the test substance (25 mg) was placed directly atop the previously moistened tissue. Length of exposition was 60 minutes. Nine tissues were used for the experiment, three per test substance (C2), three for positive control (PC) and three for negative control (NC). After rinsing, tissues were post incubated for 42 hours due to leave of damage reparation. Three hours incubation with MTT and two hours extraction period with shaking followed then. Optical density (OD570) of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues. As the test substance was suspected to be direct reducing, the test with frozen tissues was performed to detect if the test substance remaining in tissues reduces MTT directly. Direct reduction was not confirmed. Under the above-described experimental design, average viability of tissues treated by the test substance was 82.9 % of negative control average value (i.e. viability was > 50 %). The effect of test substance was negative in EpiDermTM model (the tissue was not damaged) and therefore not irritating to skin.

Eye irritation:

(1) The test substance was tested for the potential ocular corrosivity for 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 Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants, Council Regulation (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 the test substance 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.

(2) Eye irritation study with 2 female New Zealand White rabbits were exposed to 0.1 mL of test substance to one eye and the other eye served as the untreated control, according to OECD 405 and EU method B.5. A single application of the test substance to the non-irrigated eye of two rabbits produced moderate conjunctival irritation. One treated eye appeared normal at the 72-hour observation and the other treated eye appeared normal at the 7-day observation. The test substance produced individual mean scores of 0.0 for corneal opacity, 0.0 for iritis, 0.7 or 1.7 for conjunctival redness and 0.3 or 1.3 for conjunctival chemosis.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because an acute toxicity study by the dermal route does not indicate skin irritation up to the relevant limit dose level (2 000 mg/kg body weight)

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

experimental part of study performed in period from 2013-09-11 to 2013-09-16

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study from supporting substance (structural analogue)

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

water

Details on test system:

Test system
The reconstructed human epidermal model EpiDerm (EPI-200, MatTek, Ashland, USA) which consist of normal human derived epidermal keratinocytes cultured to form a multilayered highly differentiated model of the human epidermis. the EpiDerm tissues (area 0.63 cm2) are cultured on specially prepared cell culture inserts and shipped as kits.

Test procedure
a) Test substance application
The test substance (0.25 mg) was placed directly atop to the previously moistened tissue with 25 μL water. The material was spread on the tissue surface.

Procedure
On the day of experiment, EpiDerm tissues were conditioned by incubation to release transport stress related compounds and debris. After pre-incubation, tissues were topically exposed to the test chemical for 3 and 60 minutes. In each time interval three tissues were used per test chemical, three for the positive control (PC) and three for negative control (NC). After exposition, tissues were thoroughly rinsed and blotted to remove the test substance (controls).
After that, tissues were transferred to 24-well plates containing MTT medium (1 mg/mL). After 3 hour MTT incubation, the blue formazan salt formed by cellular mitochondria was extracted with 2.0 mL/tissue of isopropylalcohol and the optical density of the extracted formazan was determined using spectrophotometer Libra S22 at 570 nm. Isopropylalcohol serves as a blank.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

0.25 mg was placed directly atop to the previously moistened tissue with 25 μL water

Duration of treatment / exposure:

After pre-incubation, tissues were topically exposed to the test chemical for 3 and 60 minutes.

Number of replicates:

2

Details on test animals or test system and environmental conditions:

in vitro test on reconstructed human epidermis

Amount / concentration applied:

see Any other information on materials and methods incl. tables

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min run

Value:

109.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute treatment

Value:

76.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The testing of possible interference of test substance with test endpoint was performed. Although, after direct reduction in test-tube, there were suspicion, that the test substance was directly reducing, test in frozen tissues did not confirm this assumption. Then, the correction of results was not necessary.

As it is possible to see from the results given in Table 2, average viability of affected tissues was 109.5 % of negative control average value after 3 min treatment and 76.5 % after 60 min.
According to evaluation criteria given in Chapter 3.7 both the two values were higher than critical values (50 % and 15% respectively): 109.5 % ≥ 50 % after 3 min and 76.5 % ≥ 15 % after 60 min. The test substance should be regarded as non-corrosive.

1.1.       Direct MTT reduction- functional check in tubes

50 mgof the test substance was added to 2 mL of MTT medium. Solution was incubated for 1 hour (37±1°C, 5±1 % CO₂, moistened).

The test substance was filtered because of betterevaluation.The test substance changed colour from green to blue (see Figure 1)

Next step – test in frozen tissues - had to be done for confirmation/excluding of a direct reduction in tissues.

1.2.                 Direct MTT reduction - test in frozen tissues

The test substance (25 mg) was applied to two freeze-killed tissues for 3 min exposition and two freeze-killed tissues for 60 min

exposition. In addition, two freeze-killed tissues were treated with H₂O(for 3 min and for 60 min exposition). After 3 min and 60 min

of incubation (37±1°C, 5±1% CO₂, moistened), the test substance was rinsed and tissues were incubated with MTT solution in the same

manner as viable tissues in MTT test. Two hours extraction in isopropyl alcohol with shaking and OD measuring at 570 nm followed then.

Results are given in table 1.

Table 1:Direct MTT reduction in frozen tissues

Treatment	OD570						% NC
	tissues		mean	SD	99% confidence interval
	1	2
H2O 3 min	0.155	0.124	0.140	0.015	0.109	0.171	100.0
C1 3 min	0.162	0.144	0.153	0.009	0.135	0.171	109.7
H2O 60 min	0.107	0.080	0.094	0.014	0.067	0.121	100.0
C1 60 min	0.084	0.067	0.076	0.008	0.059	0.092	80.7

Mean OD₅₇₀value of treated tissues after 3 min treatment (0.153) fell into confidence interval OD₅₇₀of negative control (0.109-0.171) and

average OD₅₇₀value of treated tissues after 60 min treatment (0.076) fell into confidence interval OD₅₇₀of negative control (0.067-0.121),

so the test substance did not reduce MTT directly, therefore it was not necessary to correct the results of MTT test.

1.3.       MTT test

The procedure is described in chapter 3.6.3.

OD₅₇₀measuring was performed after overnight extraction. Results are given in the following table 2.

             Table 2:MTT test results (viable tissues)

time	treatment		OD570						%NC
			tissues			mean	SD	CV
			1	2	3
	NC	water	1.270	1.129	1.217	1.205	0.058	0.048	100.0
3 min	C1	Hysperse 12	1.181	1.314	1.464	1.320	0.116	0.088	109.5
	PC	8N KOH	0.265	0.225	0.338	0.276	0.047	0.170	22.9
	NC	water	1.416	1.326	1.379	1.374	0.037	0.027	100.0
60 min	C1	Hysperse 12	1.056	1.195	0.900	1.050	0.120	0.115	76.5
	PC	8N KOH	0.187	0.130	0.152	0.156	0.023	0.150	11.4

 

Notes to tables 1 and 2:

NC	negative control - solvent
PC	positive control N
C1	test substance
mean	arithmetic mean
% NC	viability of single tissues compared with negative control
SD	standard deviation calculated from individual % tissue viabilities
CV	coefficient of variance

 

Interpretation of results:

GHS criteria not met

Conclusions:

Substance is not corrosive.

Executive summary:

The test substance, was assayed for the in vitro skin corrosion in human epidermal model EpiDerm^TM. The test was performed according to Method B.40. Skin corrosion (in vitro),Council Regulation (EC) No.440/2008.

The test substance (25 mg) was placed atop the previously moistened tissue. Length of exposition was 3 and 60 minutes. Nine tissues were used for the experiment in each time, three per test substance (C1), three for positive control (PC) and three for negative control (NC). After rinsing, tissues were incubated with MTT for three hours and extracted overnight subsequently at room temperature without shaking. OD₅₇₀of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

As the test substance was suspected to be direct reducing, the test with frozen tissues was performed to detect if the test substance actually reduces MTT in tissues directly. Because this test excluded initial suspicion, the correction of MTT test results has not been made.

Under the above-described experimental design, average viability of tissues treated by the test substance, was 109.5 % of negative control average value after 3 min treatment and 76.5 % after 60 min treatment.

In the experiment arrangement given above, the test substance was non-corrosive in EpiDerm^TMmodel.

Reference 3

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental part of study performed in period: 15. 10. 2013 - 18. 10. 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study from supporting substance (structural analogue)

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

physiological saline

Details on test system:

On the day of receipt, EpiDerm tissues are conditioned by incubation to release transport stress related compounds and debris. After pre-incubations (1 and 18±3hours), tissues are topically exposed to the test chemicals for 1 hour. Three tissues are used per test
chemical and for the positive control (PC) and negative control (NC). Tissues are then thoroughly rinsed, blotted to remove the test substances, and transferred to fresh medium.
After 24 hour incubation period, the medium is replaced by fresh one. Tissues are incubated for another 18 hours. Afterwards, the MTT assay is performed by transferring the tissues to 24-well plates containing MTT medium (1 mg/ml). After 3 hour MTT incubation,
the blue formazan salt formed by cellular mitochondria is extracted with 2.0 ml/tissue of isopropanol and the optical density of the extracted formazan is determined using a spectrophotometer at 570 nm. Detailed procedure is described in SOP M/48/3 (VUOS-CETA, 2011).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test substance (25 mg) is placed directly atop to the previously moistened tissue (25 μl of PBS). The material is spread on the tissue surface.

Duration of treatment / exposure:

After pre-incubations (1 and 18±3hours), tissues are topically exposed to the test chemicals for 1 hour.

Duration of post-treatment incubation (if applicable):

24 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

82.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Under described experimental design, average viability of tissues treated by the test substance was 82.9 % of negative control average value, i. e. viability was more than 50 %.
According to the classification criteria, the test substance is considered to have no category in regard to skin irritation.

STUDY RESULTS

4.1. Direct MTT reduction-functional check in tubes

50 mg of the test substance was added to 2 ml of MTT medium. Solution was incubated for 1 hour (37±1°C, 5±1 % CO2,

moistened). The test substance was filtered because of better evaluation. The test substance changed colour from green to

blue.

Next step – test in frozen tissues -had to be done for confirmation/excluding of a direct reduction in tissues.

4.2. Direct MTT reduction -MTT test in frozen tissues

The test substance (25 mg) was applied to two freeze-killed tissues for 60 min exposition. In addition, two freeze-killed

tissues were treated with PBS (negative control). After 60 min of incubation (37±1°C, 5±1% CO2, moistened), the test

substance was rinsed and tissues were incubated with MTT solution in the same manner as viable tissues in MTT test.

Two hours extraction in isopropyl alcohol with shaking and OD measuring at 570 nm followed then.

Results are given on Table 1.

Table 1: Direct MTT reduction in frozen tissues

 

	OD570					% NC
	tissues		Mean	SD	99% confidence interval
	1	2

PBS 60 min	0.089	0.080	0.085	0.005	0.075	0.094	100.0
C2 60 min	0.080	0.056	0.068	0.012	0.044	0.092	80.5

 

Mean average OD570value of treated tissues after 60 min treatment (0.068) was lower than average OD570value of negative

control (0.085), so the test substance did not reduce MTT directly, therefore it was not necessary to correct the results of MTT test.

4.3. MTT test

The test substance (25 mg) was placed directly atop to the tissue previously moistened with 25 µl of PBS. The material was

then spread on the tissue surface. Tissues were exposed to the test chemical for 1 hour. After exposition, tissues were

thoroughly rinsed and blotted to remove the test substance. Then, tissues were let to post-incubate for 24 (medium exchange)

+ 18 hours (37±1°C, 5±1% CO2, moistened).

Afterwards, the MTT assay was performed by transferring the tissues to 24-well plates containing MTT medium (1 mg/ml).

After 3 hour MTT incubation, the blue formazan salt formed by cellular mitochondria was extracted with 2.0 ml/tissue of

isopropyl alcohol and the optical density of the extracted formazan was determined using a spectrophotometer at 570 nm.

OD570measuring was performed after 2-hour extraction with shaking. Results are given in the Table 2. Extracts as well as

tissues after extraction are given in Figure 2.

Table 2:OD570values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities

Treatment	Parameter				OD570				Mean	SD	Average viability (% NC)
			1	2		3
NC		OD570	1.908	1.809		1.938		1.885		0.055
(PBS)		viability	101.2	96.0		102.8		100.0		2.9	100.0
C2		OD570	1.668	1.524		1.498		1.563		0.075
(234/13)		viability	88.5	80.8		79.5		82.9		4.0	82.9
PC		OD570	0.054	0.059		0.061		0.058		0.003
(5% SDS)		viability	2.9	3.1		3.2		3.1		0.2	3.1

 

Notes:

NC              negative control

PC              positive control

C2              test substance

mean           arithmetic mean

SD              standard deviation calculated from individual % tissue viabilities

viability (% NC) viability of single tissues compared with negative control

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test substance considered not irritating to skin.

Executive summary:

Test substance, was assayed for the in vitro skin irritation in human epidermal model EpiDermTM. The test was performed according to Method B.46. In vitro skin irritation: Reconstructed human epidermis model test and Protocol for: In Vitro EpiDermTM Skin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT.

After pre-incubation of tissues, the test substance (25 mg) was placed directly atop the previously moistened tissue. Length of exposition was 60 minutes. Nine tissues were used for the experiment, three per test substance (C2), three for positive control (PC) and three for negative control (NC).

After rinsing, tissues were post incubated for 42 hours due to leave of damage reparation. Three hours incubation with MTT and two hours extraction period with shaking followed then. Optical density (OD570) of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

As the test substance was suspected to be direct reducing, the test with frozen tissues was performed to detect if the test substance remaining in tissues reduces MTT directly. Direct reduction was not confirmed.

Under the above-described experimental design, average viability of tissues treated by the test substance was 82.9 % of negative control average value (i.e. viability was > 50 %).

The effect of test substance was negative in EpiDermTM model (the tissue was not damaged).

According to the classification criteria given in chapter 3.9. of this report (see Method B.46. In vitro skin irritation: Reconstructed human epidermis model test. Commission Regulation (EC) No. 761/2009, 23rd July 2009; 2.2. Interpretation of results, L 220/29), the test substance is considered to have no category in regard to skin irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study from supporting substance (structural analogue)

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)

Species:

cattle

Strain:

not specified

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.

Vehicle:

water

Controls:

yes, concurrent positive control

yes, concurrent negative control

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.

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

30.06

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

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
Test material	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
Test material	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 Test material – 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.

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, 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 Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants, Council Regulation (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 (IVIS) was calculated from the values of opacity and permeability.

The In Vitro Irritancy Score (IVIS) for the test material 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.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 May 2017 to 18 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Age at study initiation: 12 to 52 weeks old
- Weight at study initiation: 3.34 or 3.54 kg
- Housing: Individually in suspended cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23°C
- Humidity (%): 70%
- Air changes (per hr): 15/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 4 May 2017 To: 18 May 2017

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL of the test substance, which was found to weigh 95mg

Duration of treatment / exposure:

Single exposure

Observation period (in vivo):

Assessment of ocular damage/irritation was made at 1, 24, 48, and 72 hours.

Number of animals or in vitro replicates:

2

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): None
- Time after start of exposure: Not applicable

SCORING SYSTEM: Draize

TOOL USED TO ASSESS SCORE: Ophthalmoscope

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

conjunctival redness

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.7

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.7

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible

Irritant / corrosive response data:

A single application of the test substance to the non-irrigated eye of two rabbits produced moderate conjunctival irritation. One treated eye appeared normal at the 72-hour observation and the other treated eye appearred normal at the 7-day observation.

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance produced individual mean scores of 0.0 for corneal opacity, 0.0 for iritis, 0.7 or 1.7 for conjunctival redness and 0.3 or 1.3 for conjunctival chemosis.

Executive summary:

Eye irritation study with 2 female New Zealand White rabbits were exposed to 0.1 mL of test substance to one eye and the other eye served as the untreated control, according to OECD 405 and EU method B.5. A single application of the test substance to the non-irrigated eye of two rabbits produced moderate conjunctival irritation. One treated eye appeared normal at the 72-hour observation and the other treated eye appearred normal at the 7-day observation. The test substance produced individual mean scores of 0.0 for corneal opacity, 0.0 for iritis, 0.7 or 1.7 for conjunctival redness and 0.3 or 1.3 for conjunctival chemosis.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available