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

Skin irritation / corrosion

Administrative data

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)

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

In vitro test system

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

Test animals

Details on test animals or test system and environmental conditions:

in vitro test on reconstructed human epidermis

Test system

Amount / concentration applied:

see Any other information on materials and methods incl. tables

Results and discussion

In vitro

Results

Results 1

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

Results 2

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.

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:

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.