Polychloro copper phthalocyanine

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 national standard methods

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:

no

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
- Mass-specific surface area (BET): 69 m²/g
- TEM (primary particle diameter): 39 nm

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:

The biological oxidative damage is given in trolox-equivalent units (TEU) per surface dose of particles.


sBOD (test substance) = 13 ± 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

The test substance generate a weak oxidative damage that is measurable above the error bar of the negative control. However, the oxidative damage remains below the positive control and therefore it is categorized as passive by the FRAS assay.

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

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

Principles of method if other than guideline:

OH radicals generated by the particle surface are stabilized by converting the spin trap DMPO to DMPO-OH during an incubation and reaction time of 30 minutes. The reaction occurs within the ESR capillary and does not require further sample handling. The characteristic four-peak spin signal of DMPO-OH is then detected by ESR and is evaluated as peak-to-peak signal, alternatively directly converted to a spin concentration.

GLP compliance:

no

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
- Mass-specific surface area (BET): 69 m²/g
- TEM (primary particle diameter): 39 nm

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), then n-octanol, finally methanol. 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
-negative control: BaSO4

Positive control:

CuO (partially soluble)

Details on results:

The test substance generated a reactivity signal that is statistically different from the blank control (0.8 +/- 0.3*10^12 spins/mL vs. 1.2*10^12 spins/mL for the blank control). Therefore, the results indicate borderline surface reactivity and is thus not considered as reactive.

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.

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
- Name as cited in the publication: CuPhthalo_halogen
- In the publication a wrong CAS number (CAS 132-53-5) is noted.




TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
Due to poor dispersibility, the test material was pre-wetted in 0.5 % (v/v) ethanol. For this purpose, a stock dispersion of 2.56 mg/mL (with the further addition of 0.05 % (w/v) bovine serum albumin to warrant dispersion) was prepared by 10-min bath ultrasonication. Then the necessary amount of fluid (cell culture medium F-12K or Ringer phosphate glucose (KRPG) buffer for H2O2 determination) was added to achieve final maximum concentrations of
180 μg/mL F-12K medium or 360 μg/mL KRPG buffer, the latter being diluted twofold under testing conditions. Upon suspension and/or dilution, the tubes containing the test material were briefly vortexed to remove particles from the vessel wall. Afterwards, the preparations were ultrasonicated for 10 s. All test material suspensions were prepared shortly before in vitro application.
- Final dilution of a dissolved solid, stock liquid or gel:
For LDH, GLU and TNF-α determination, the suspensions were serially diluted with F-12K medium to achieve test concentrations of 22.5, 45, 90, and 180 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)

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 h. For the determination of LDH, GLU, and TNF-α release, cell culture supernatants were sampled after 16 h of incubation.

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.
- 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 elicited dose-dependent increases of LDH, GLU and TNF-α with significant LOAECs of 12420 mm²/mL, each.
Accordingly, two parameters had LOAECs above the threshold value of 6000 mm²/mL. Therefore, the test substance is classified as passive.

Description of key information

Two abiotic methods were combined to examine the potential of the the test substance 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.

In the FRAS as well as in the EPR assay the test substance was considered as passive.

The test on alveolar macrophages confirmed the "passive" outcome of the abiotic tests.