Low chlorinated copper, [29H,31Hphthalocyaninato(2-)-N29,N30,N31,N32]-, wherein the number of chlorines is more than or equal to 0 and less than or equal to 7

Specific investigations: other studies

Administrative data

Endpoint:

biochemical or cellular interactions

Remarks:

in vitro alveolar macrophage assay

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test material was incubated with Rat NR8383 alveolar macrophages in protein-free culture medium. Lactate dehydrogenase, glucuronidase, and tumour necrosis factor alpha were assessed after 16 h. In parallel, H2O2 was assessed after 1.5 h.

GLP compliance:

not specified

Type of method:

in vitro

Endpoint addressed:

other: membrane disruption and activation of alveolar macrophages

Test material

Test material information

Constituent 1

Test material form:

solid: nanoform

Details on test material:

- Name of test substance: Tetrachlioro-Cu-Phth (3-4 Cl atoms)
- Chemical Name: Low chlorinated copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, wherein the number of chlorines is more than or equal to 0 and less than or equal to 7
- Batch identification: 190020P040
- Date of production: 22 Jun 2019
- Storage conditions: room temperature
- Physical state/ appearance: solid blue
- Water solubility: insoluble
- Particle size distribution (TEM): = 46.0 nm (D50)
- Mass median aerodynamic diameter (MMAD): 1.93 – 2.98 µm
- Geometric standard deviation (GSD): 2.67 – 3.13
- Shape of particles: spherical
- Aspect ratio: 1.3:1
- Surface area of particles (BET): 17.2 m^2/g
- Crystal structure: crystalline
- Coating: no
- Surface properties: not applicable
- Density: 1726 kg/m^3 at 20°C
- Moisture content: not specified
- Residual solvent: not specified
- Activation: not applicable
- Stabilisation: not applicable

Specific details on test material used for the study:

Physical state/ appearance: solid blue
- Purity: ca 100 %

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Particles were dispersed according to the NanoGenotox protocol which uses small amounts of serum albumin to stabilize non-polar particles: A total of 15.36 mg of the dry powder was weighed into 20 mL glass vials, wetted with 30 μL ethanol, then mixed with 6 mL double distilled water containing 0.05% bovine serum albumin. Thus, the stock suspension contained 2.56 mg particles/mL, 0.5% (vol/vol) ethanol, and 0.05% bovine serum albumin. The stock suspension was ultrasonicated for 16 min with a Branson 450D sonifier. For experiments the stock suspension was mixed with one volume of double concentrated KRPG buffer or double concentrated F12-K medium, to achieve a physiological medium composition needed for the testing of cytotoxicity and cellular H2O2 generation, respectively. The resulting suspension was serially diluted in serum-free F12-K medium to obtain concentrations of 180, 90, 45 and 22.5 μg/mL. The suspension was also serially diluted in KRPG buffer, first to obtain 360, 180, 90, and 45 μg/mL; 100 μL of these suspensions were then added to cells covered with 100 μL of KRPG, to achieve the final test concentrations of 180, 90, 45 and 22.5 μg/mL.

Test animals

Species:

other: NR8383 cells (alveolar macrophage cell line derived from rat lung lavage cells)

Administration / exposure

Vehicle:

other: F-12K medium and KRPG buffer (depending on the respective investigation)

Duration of treatment / exposure:

16 h (for the determination of LDH, GLU, and TNF-α release); 1.5 h (for the determination of H2O2 formation)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

22.5 other: µg/mL

Doses / concentrations 2

Dose / conc.:

45 other: µg/mL

Doses / concentrations 3

Dose / conc.:

90 other: µg/mL

Doses / concentrations 4

Dose / conc.:

180 other: µg/mL

Details on study design:

- Rat NR8383 cells, routinely cultured in F-12K medium supplemented with 2 mM glutamine, penicillin/streptomycin (100 U/10 mg/mL) and 15 % (v/v) fetal calf serum in 500 mL flasks under standard cell culture conditions (37 °C; 5 % CO2) and passaged once a week, were detached from the substrate by mechanical agitation, dispersed by pipetting, seeded into 96-well plates at 3 × 10^5 live cells per well and incubated in F-12K medium supplemented with 5 % FCS for 24 h. For test material application, supernatants were withdrawn, and test material-containing phenol red-free F-12K medium, supplemented with 2 mM glutamine and 100 U/100 μg/mL penicillin/streptomycin, was applied onto the cells.
- To correct for test material-specific adsorption and/or scattering of light, cell-free test material-containing controls were included in all test runs for all dilution steps.
- Cells were incubated with the test substance for 16 or 1.5 h. For the determination of LDH, GLU, and TNF-α release, cell culture supernatants were sampled after 16 h of incubation. In a parallel approach, supernatants were sampled after 1.5 h of incubation to assess H2O2 formation.

The stock solution of the test material prepared in F-12K medium was tested for contamination with viable bacteria and/or fungi. For this purpose, 50 μl of the aqueous suspension was plated onto a conventional maltose and a casein peptone agar. Plates were incubated at 37°C for 72 h and inspected for colonies of microorganisms after 24, 48 and 72 h.

Composition of KRPG-buffer was NaCl (129 mM), KCl (4.86 mM), CaCl2 (1.22 mM), NaH2PO4 (15.8 mM), glucose (5.5 mM), pH 7.3-7.4.

Examinations

Examinations:

- Parameters examined: cellular release of lactate dehydrogenase (LDH), β-glucuronidase (GLU) as indicators of membran disruption and macrophage activation; bioactive TNF-α as indicator of pro-inflammatory reactions; H2O2 as indicator of oxidative stress induction
- The lowest concentration at which significant effects were recorded for a given endpoint-specific test result was termed in vitro LOAEC.
- To convert the mass-based test material concentrations into surface area-based concentrations (mm²/mL), the applied mass concentrations (μg/mL) were multiplied with the respective test material’s surface area (m²/g).

Positive control:

Quartz DQ12

Corundum (AL2O3) served as negative control

Results and discussion

Details on results:

The gravitationally settled fraction of Tetrachlioro-Cu-Phth (3-4 Cl atoms) up to a concentration of 180 μg/mL was largely ingested by alveolar macrophages (NR8383 cells), although many small particles were not engulfed at the highest concentration. This ears the risk of underestimation of effects
There were dose-dependent increases of LDH, H2O2, TNFα, and GLU release which became significant upon 90 and 180 μg/mL, respectively. Overall, according to the criteria published by Wiemann et al 2016 and taking into account the BET of 17.2 m2/g, the pigment is classified as active.

Any other information on results incl. tables

Table 1: Hydrodynamic diameter (nm) of the test material at 18 mg/L in H2O, KRPG and F-12K-medium after dispersion according to the NanoGenotox protocol

	size (mean)		size (mode)		size (D10)		size (D50)		size (D90)
	average	SEM	average	SEM	average	SEM	average	SEM	average	SEM
H20	165	2.8	140.7	1.5	83.6	2.2	143.1	2.4	252.7	7.6
F-12K	174.9	4.6	148.9	4.6	93.7	6.6	155.8	4.3	267.7	4.2
KRPG	176.9	5.8	138.4	5.4	93.3	6.4	160.7	8.3	271.3	5

Under cell culture conditions, i.e. in F-12K medium, the H2O dispersed test material hardly changed its hydrodynamic diameter (Table 1). Importantly, there was a layer of micron-sized agglomerates/aggregates at the bottom of the cell culture vials (see Figure). The size of these aggregates/agglomerates was mostly <5 μm (Figure, upper panels) with some larger particles up to 10 μm. The density of settled agglomerates, which are not included in the PTA measurements, correlated with the administered concentration (Figure). Settled aggregates/agglomerates were largely engulfed by NR8383 cells most many of which contained colored inclusions (Figure). Nevertheless, a considerable number of small particles were still visible among cells after 16 h of incubation, indicating incomplete particle uptake.

In vitro toxicity data

Control cells reacted as expected: non-particle treated, or LPS-treated cells (a control for TNF induction) were undamaged. Corundum treated cells were particle-laden but undamaged. Quartz DQ12 treated cells were particle laden and appeared granular and partly deteriorated.

NR8383 cells exposed to the test material nearly completely cleared the settled fraction of particles from the bottom of the culture wells up to a concentration of 180 μg/mL (see Figure, bottom).

Effects on the release of lactate dehydrogenase (LDH), glucuronidase (GLU), H2O2 and TNFα are shown in the figure and table 2.

Table 2: In vitro effects of Tetrachlioro-Cu-Phth (3-4 Cl atoms) on NR8383 macrophages in comparison to corundum and quartz DQ12 (n=3).

	[μg/mL]	LDH	GLU			H2O2			TNFα
LDH	[μg/mL]	[% of pos. CTR]	[% of pos. CTR]			[μmol/L]			[pg/mL]
Tetrachlioro-Cu-Phth (3-4 Cl atoms)	0	19.1 ± 1.5	2.1	±	0.8	1.0	±	0.2	4.8	± 2.2
	22.5	19.0 ± 2.1	1.8	±	0.7	1.1	±	0.1	3.8	± 2.9
	45	25.8 ± 2.4	2.5	±	0.7	1.2	±	0.6	6.3	± 3.4
	90	44.0 ± 5.4***	4.8	±	1.1	1.7	±	0.4**	15.2	± 2.8       ***
	180	89.0 ± 2.7***	15.3	±	0.2***	2.1	±	1.0***	35.5	± 1.6       ***
Corundum	0	20.5 ± 2.0	2.3	±	0.2	0.5	±	0.0	5.2	± 1.4
	22.5	17.6 ± 2.8	2.9	±	0.8	0.7	±	0.0	5.5	± 2.3
	45	23.2 ± 1.6	3.6	±	1.1	0.9	±	0.2	6.1	± 1.9
	90	25.9 ± 2.6	3.9	±	1.2	1.5	±	0.7	6.5	± 2.2
	180	30.2 ± 2.9	3.9	±	0.9	2.8	±	0.7**	6.5	± 1.8
Quartz DQ12	0	20.5 ± 2.0	2.3	±	0.2	0.5	±	0.0	5.2	± 1.4
	22.5	19.0 ± 3.0	1.9	±	0.1	0.8	±	0.1	5.9	± 1.5
	45	31.6 ± 4.8*	4.2	±	1.2	1.0	±	0.1	12.5	± 1.8     **
	90	79.9 ± 6.2***	14.7	±	3.2***	1.8	±	0.4	44.4	± 5.0     ***
	180	120.1 ± 23.0***	31.5	±	10.3***	2.7	±	0.8**	61.5	± 9.7     ***
Zymosan	360					18.4	±	0.8
LPS	0.5								245.5	± 75.4