Copper phtalocyanine monosulfonated and disulfonated

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was performed according to internationally valid test method in accordance 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:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

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 disuphonated

IUPAC Name:

Copper phtalocyanine mono- and disuphonated

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
- Molecular formula (if other than submission substance): C32H15N8Cu(SO3H)1.4x2.5 H2O
- Molecular weight (if other than submission substance): 733.3
- Smiles notation (if other than submission substance):
- InChl (if other than submission substance):
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type: metall-organic complex
- Physical state: powder
- Analytical purity: 99.5 % w/w
- Impurities (identity and concentrations):water soluble salts 0.5 %w/w
- Composition of test material, percentage of components:
- Isomers composition:
- Purity test date:
- Lot/batch No.: 01
- Expiration date of the lot/batch:
- Radiochemical purity (if radiolabelling):
- Specific activity (if radiolabelling):
- Locations of the label (if radiolabelling):
- Expiration date of radiochemical substance (if radiolabelling):
- Stability under test conditions: Unlisted.
- Storage condition of test material:
- Other:

Test animals

Details on test animals or test system and environmental conditions:

in vitro test on reconstructed human epidermis

Test system

Type of coverage:

other: not relevant

Preparation of test site:

other: not relevant

Vehicle:

other: water

Amount / concentration applied:

see Any other information on materials and methods incl. tables

Details on study design:

see Any other information on materials and methods incl. tables

Results and discussion

In vivo

Irritant / corrosive response data:

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.

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

Interpretation of results:

other: non corrosive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

substance is not corrosive

Executive summary:

Test substanceHysperse 12was assayed for thein vitroskin corrosion in human epidermal model EpiDerm^TM. The test was performed according toMethod B.40. Skin corrosion (in vitro),Council Regulation (EC) No.440/2008.

           The test substance (25 mg)wasplaced atop thepreviously moistenedtissue.Length of exposition was 3 and60 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 substanceHysperse 12was109.5%of negative control average value after 3 min treatment and76.5 %after 60 min treatment.

In the experiment arrangement given above, the test substanceHysperse 12was non-corrosive in EpiDerm^TMmodel.