29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32 copper

Specific investigations: other studies

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

specific investigations: other studies

Remarks:

Surface reactivity (abiotic)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

equivalent or similar to guideline

Guideline:

other: ISO TS 18827:2017 Nanotechnologies

Version / remarks:

Electron spin resonance (ESR) as a method for measuring reactive oxygen species (ROS) generated by metal oxide nanomaterials

GLP compliance:

not specified

Type of method:

other: in chemico

Endpoint addressed:

other: surface reactivity (abiotic)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material:
130114P050
- Purity: 96.62 %
- Physical state: solid, blue
- Mass-specific surface area (BET): 58 m²/g
- TEM (primary particle diameter): 17 nm (D50)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Preliminary purification step (if any):
To avoid false positive results (detection of additives, impurities etc.) pigments were purified by sequential solvent washes: first methanol/toluene (80/20, Merck, HPLC grade), then n-octanol (Sigma-Aldrich, Spectrophotometric grade), finally methanol (Merck, HPLC grade). For each solvent the pigment was shaken for 2 hours at room temperature, recovered by centrifugal pelleting (20,000 rpm, 1h), and dried under vacuum (1.3 mbar, 90 °C, 1h).

Duration of treatment / exposure:

30 min

No. of animals per sex per dose:

- replicates: triplicate

Details on study design:

- Amount of test item / trial: 10 mg
- Medium used (composition): DMPO spin trap in phosphate buffer
- Incubation time, temperature: 30 min at room temperature
- Blank: DMPO spin trap in phosphate buffer

Examinations:

- Detection method: Electron Paramagnetic Resonance Spectroscopy (EPR)
- Metric: relative increase of biological oxidative damage (sBOD) against negative control (x-fold control) at a nanomaterial concentration of 1 m² /mL

Positive control:

CuO (partially soluble)

Details on results:

The test substance generated a reactivity signal that is statistically different from the blank control (9.5*10^12 spins/mL vs. 1.2*10^12 spins/mL for the blank control). Therefore, the results indicate borderline surface reactivity.

Reference 2

Endpoint:

specific investigations: other studies

Remarks:

Surface reactivity (abiotic)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Ferric Reduction Ability of Serum (FRAS) assay measures the surface reactivity of nanomaterials (NMs) under physiological conditions. The test material NMs are incubated with human blood serum (HBS). The antioxidative components in the serum serve as reporter molecules as they are consumed during incubation with the NMs. Afterwards, the NMs are centrifuged and the supernatant human blood serum is incubated in the FRAS reaction with Fe3+. The reduction of Fe3+ to Fe2+ is detected optically by a color change from transparent to blue. A high consumption of antioxidative components will be observed in a reduction of the blue color.

GLP compliance:

not specified

Type of method:

other: in chemico

Endpoint addressed:

other: surface reactivity (abiotic)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 130114P050
- Purity: 96.62 %
- Physical state: solid, blue
- Mass-specific surface area (BET): 58 m²/g
- TEM (primary particle diameter): 17 nm (D50)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Preliminary purification step (if any): To avoid false positive results (detection of additives, impurities etc.) pigments were purified by sequential solvent washes: first methanol/toluene (80/20, Merck, HPLC grade), then n-octanol (Sigma-Aldrich, Spectrophotometric grade), finally methanol (Merck, HPLC grade). For each solvent the pigment was shaken for 2 hours at room temperature, recovered by centrifugal pelleting (20,000 rpm, 1h), and dried under vacuum (1.3 mbar, 90 °C, 1h).

Route of administration:

other: test material is in direct contact to human serum

Duration of treatment / exposure:

180 min

Frequency of treatment:

one treatment

No. of animals per sex per dose:

- replicates: triplicate

Details on study design:

- 10 mg of test item applied to 100% human serum (Sigma Aldrich, P2918-100mL) at 37°C for 180 min
- the sample and serum are centrifuged at 14,000 g (11,900 rpm in specific centrifuge) for 150 minutes
- the FRAS reagents were handled in the dark
- calibration of the FRAS absorption signal with a concentration series of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a water-soluble analogue of vitamin E
- Blank: pure serum

Examinations:

- measurement of UV/VIS absorption at 593 nm
- Metric: Surface-based biological oxidative damage (sBOD) in nM Trolox-equivalent units (TEU) per m² of nanomaterial surface = nM TEU / m² , at a nanomaterial concentration of 1 m² /mL.

Positive control:

Mn2O3 (insoluble), CuO (partially soluble)

Details on results:

sBOD (test substance) = 2.5 ± 0.3 nmol TEU / m²
sBOD (Mn2O3, positive control) = 4742 ± 27 nmol TEU / m²
sBOD (CuO, positive control) = 9586 ± 29 nmol TEU / m²
sBOD (negative control, no particles) = 2 ± 0.7 nmol TEU / m²

Based on the assessment criteria, the test substance is regarded as "passive". However, small particles remained suspended in the serum after separation. As interferences were anticipated, the test was repeated by the complementary technique EPR.

Reference 3

Endpoint:

biochemical or cellular interactions

Remarks:

in vitro alveolar macrophage assay

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Colours and Effects GmbH
-solid, blue
- Purity: ≥ 99 %

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.

Species:

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

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)

Dose / conc.:

22.5 other: µg/mL

Dose / conc.:

45 other: µg/mL

Dose / conc.:

90 other: µg/mL

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:

- 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

Details on results:

The test substance did not elicit a dose-dependent increase of LDH and GLU. With respect to the H2O2 activity measurement, there was some optical interference due to the color of the suspension. This interference occurred progressively from 22.5 to 180 μg/mL, was observed in three independent repetitions, and led to negative values, which were not considered for the final evaluation.
Overall, the test substance is classified as passive.

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
H2O	108.2	2.7	70.9	2.5	60.2	2	87.4	3.2	166.5	2.9
F-12K	127	7.6	91.2	19.8	66.5	5.5	114.5	9	191.2	7.1
KRPG	128.2	2.8	73.9	7.1	63.7	2.7	117.3	3.6	189	5.7

Under cell culture conditions (in F-12K medium), the H2O dispersed material showed a slight increase (approximately +17.3 % of the mean value) in hydrodynamic diameter (Table 1) indicating moderate particle agglomeration. Importantly, there was a layer of micron-sized agglomerates/aggregates at the bottom of the cell culture vials, which were not included in the PTA measurements. The density of settled agglomerates correlated with the administered concentration (Figure 4). Although the size of single aggregates/agglomerates reached up to 25 μm (Figure 4, upper panels), most of the settled material was engulfed by NR8383 cells. Only at the highest concentration (180 μg/mL), a few small particles remained visible among the cells after 16 h.

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 cleared the settled fraction of particles from the bottom of the culture wells nearly completely up to a concentration of 180 μg/mL.

The test material elicited no significant effects on the release of LDH, GLU, TNFα, and H2O2 from NR8383 macrophages up to a concentration of 180 μg/mL (see attached table 2). With respect to the H2O2 activity measurement there was some optical interference due to the color of the suspension. This interference occurred progressively from 22.5 to 180 μg/mL, was observed in three independent repetitions, and led to negative values which were not considered for the final evaluation.

Additional information

Two abiotic methods were combined to examine the potential of the (mono)chlorinated form of the test substance (CAS 12239-87-1) to induce biological oxidative damage. The FRAS assay (Ferric Reduction Ability of Serum) uses antioxidative components present in human blood serum as reporter molecules. The ability of the pre-incubated human blood serum to reduce Fe3+ to Fe2+ is then detected optically by a color change from transparent to blue. The EPR assay, the second method, does not require separation of the organic pigments from the incubation medium. Under consideration of the respective specific surface, the test material is then classified as “active” or “passive” to induce oxidative damage.

Whereas the FRAS assay with simple centrifugation indicated no relevant reactivity, the EPR assay indicated borderline reactivity for the test substance.

This test item was also examined in a cell-based surface reactivity assay and in a short-term inhalation study (key study in chapter 7.5.2 of the registration dossier) to verify the obtained results. Whereas the test on alveolar macrophages confirmed the outcome of the abiotic tests (Wiemann et al. 2016) the short-term inhalation study did not reveal any adverse histopathological findings and no treatment related changes in BALF up to and including the highest tested concentration of 30 mg/m³ air.