Bis(N,N-dimethylpropane-1,3-diamine-N)[29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]cobalt(1+) chloride

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-11-07 to 2016-12-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

06-Jul-2012

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Bis(N,N-dimethylpropane-1,3-diamine-N)[29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]cobalt(1+) chloride

EC Number:

284-943-5

EC Name:

Bis(N,N-dimethylpropane-1,3-diamine-N)[29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]cobalt(1+) chloride

Cas Number:

84989-53-7

Molecular formula:

C42H44CoN12.Cl

IUPAC Name:

hydroxylamine

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human-derived epidermal keratinocytes

Source strain:

not specified

Vehicle:

other: DPBS

Remarks:

to improve the contact between the test item and the epidermis, DPBS was used

Details on test system:

- Source: MatTek Corporation (82105 Bratislava, Slovakia)
- The EpiDerm™ tissue: normal, human-derived epidermal keratinocytes which have been cultured to
form a multilayered, highly differentiated model of the human epidermis
- Surface: 0.63 cm2
- Pre-incubation: 60 minutes in the incubator (37 ± 1 °C, 5% CO2). Then transferred to new wells for about 24 ± 2 hours (37 ± 1.5 °C, 5 ± 1%
CO2, 95 ± 5% RH).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg test item + 25 µL DPBS

Duration of treatment / exposure:

60 min.

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Test system

Details on study design:

Details of the test procedure used
- EpiDerm™ tissue of human-derived epidermal keratinocytes was used (EPI-200-SIT Kit, Lot No.: 23378 Kit E, 23385)
- Conditions of exposure: 37 ± 1 C°C, 5 % CO2, 95% RH
- Washing: inserts gently rinsed with DPBS at least 15 times
- Number of tissue replicates used per test chemical and controls: 3
- MTT assay: incubation with 1 mL of MTT solution for 60 minutes at 37 ± 1 °C

- Data evaluation: the following was calculated: The mean OD of the three negative control tissues was calculated after blank correction. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:
Relative viability(%) = (mean OD test item / positive control / mean OD negative control) x 100.
For the test item and the positive control the mean relative viability ± rel. standard deviation of the three individual tissues was calculated

- Description of evaluation criteria: The test substance may be considered as non-irritant to skin in accordance with UN GHS “No Category” if the tissue viability after exposure and post-treatment incubation is more than 50%.
- Historical data positive control: Mean Viability: 3.9%; Rel. Standard Deviation: 4.4%; Mean Absorption: 1.831

- Acceptability of the Assay: the results are acceptable if
(1) tissue viability is meeting the acceptance criterion, i.e. if the mean OD570 of the negative control tissues is ≥ 0.8 and ≤ 2.8 (negative control),
(2) the relative tissue viability of the positive control is ≤ 20% (positive control) and
(3) the SD of 3 identical replicates is < 18%.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Single test with three tissues

Value:

>= 88.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The acceptance criteria were met

Any other information on results incl. tables

Pre-experiments:

The MTT-reducing capacity was tested. The mixture turned blue/purple.

The non-specific MTT reducing capacity (NSMTT) was assessed. The NSMTT was 8.2 % (≤ 30 %), hence correction is needed.

The non-specific coloring was also assessed. The NSC was found to be 2.1 % (≤ 5 %). Therefore, no correction of the results was necessary.

Main test:

Name	Negative control			Positive Control			Test Material
Tissue	1	2	3	1	2	3	1	2	3
absolute OD570	2.021	1.928	1.971	0.105	0.107	0.113	1.927	1.822	2.005
	2.020	1.959	2.045	0.114	0.110	0.118	1.995	1.824	2.022
OD570(blank-corrected)	1.978	1.885	1.928	0.062	0.064	0.070	1.884	1.779	1.962
	1.977	1.916	2.002	0.071	0.067	0.075	1.952	1.782	1.979
mean OD570of the duplicates (blank-corrected)	1.978	1.900	1.965	0.067	0.066	0.073	1.918	1.780	1.970
total mean OD570of 3 replicate tissues (blank-corrected)	1.948^*			0.068			1.890
TODTT	-			-			1.808
SD OD570	0.042			0.004			0.098
relative tissue viability [%]	101.5	97.6	100.9	3.4	3.4	3.7	98.5	91.4	101.2
mean relative tissue viability [%]	100.0			3.5			97.0
NSMTT-corrected mean relative tissue viability [%]	-			-			88.8
SD tissue viability [%]	2.1			0.2			5.0
CV [% viabilities]	2.1			5.5			5.2

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The mean tissue viability was 88.8% (NSMTT corrected). Since this is above 50%, it is concluded that the test item shows no irritant effects.

Executive summary:

The potential of the test item to induce skin irritation was analysed by using the three-dimensional human epidermis model EpiDerm (MatTek) comprising a reconstructed epidermis with a functional stratum corneum. The test was according to OECD 439 guideline and in compliance to GLP.

The test substance was applied topically to the EpiDerm tissue for 60 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS.

The test item showed MTT-reducing capability. Since the substance was classified as “non-irritant” in the main experiment, a correction procedure for determination of NSMTT (non-specific reduction of MTT) was necessary. The true MTT metabolic conversion (TOD_TT) of the test item treated living tissues TM was therefore corrected to 1.808, NSMTT was calculated to 8.2%.

The test item showed colouring potential after mixture with water and isopropanol. The spectral analysis of the mixture showed relevant absorption in the range of 570 ± 30 nm. Since the substance was classified as “non-irritant” in the main experiment (mean relative tissue viability > 50%), a correction procedure for determination of NSC_living (non-specific colour of additional viable tissues) was necessary. NSC_livingwas 2.1 % (≤ 5%) relative to the negative control of living epidermis, therefore no correction of the results and no determination of NSC_killed ( non-specific colour of additional killed tissues) was necessary.

The mean relative tissue viability (% negative control) was 88.8% (NSMTT-corrected) after 60 min treatment and 42 h post-incubation. Since this is > 50% it can be concluded that the test item showed no irritant effects.

The controls confirmed the validity of the study.