6,15-dihydroanthrazine-5,9,14,18-tetrone

Specific investigations: other studies

Link to relevant study record(s)

Reference

Endpoint:

cell culture study

Remarks:

macrophages

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline available

Principles of method if other than guideline:

Activation and/or damage of rat alveolar macrophages: four parameters are tested in the cell culture supernatant under serum-free conditions: H2O2, tumour necrosis factor alpha (TNF), lactate dehydrogenase (LDH), and the lytic enzyme glucuronidase.

A particle is classified as to be active if the Low Observed Adverse Effect Level (LOAEL) multiplied by the specific BET value drops below the threshold value of 6000 mm2/mL for at least 2 out of the 4 tests. The derivatization of this criterion is outlined in the study of Wiemann et al. 2016 (J. Nanobiotechnology 14:16) where the outcome of 18 short term inhalation studies on nanomaterials are compared with macrophage testing results

GLP compliance:

no

Type of method:

in vitro

Endpoint addressed:

repeated dose toxicity: inhalation

Specific details on test material used for the study:

Batch identification: P 112045

Species:

other: rat alveolar macrophages (cell line NR8383)

Details on test animals or test system and environmental conditions:

Information from ATCC (https://www.atcc.org/en/Products/All/CRL-2192.aspx#characteristics):
NR8383 (normal rat, August 3, 1983) was established from normal rat alveolar macrophage cells obtained by lung lavage. The cells were cultured in the presence of gerbil lung cell conditioned medium for approximately 8 to 9 months.
Subsequently the requirement for exogenous growth factors was lost.
The cells exhibit characteristics of macrophage cells.
Phagocytosis of zymosan and Pseudomonas aeruginosa, nonspecific esterase activity, Fc receptors, oxidative burst,IL-1, TNF beta and IL-6 secretion, and replicative response to exogenous growth factors.
The cells respond to appropriate microbial, particulate or soluble stimuli with phagocytosis and killing.
NR8383 cells respond to bleomycin by secreting latent transforming growth factor (TGF beta).
Stimulation with bleomycin also increases TGF beta mRNA expression.
These cells are sensitive to endotoxin.
LPS levels of 1 to 10 ng/mL inhibit replication by 50%.
LPS inhibition is nontoxic and reversible even after levels up to 0.001 mg/mL for extended periods.


The alveolar macrophage cell line NR8383 (ATCC) was cultured according to ATCC guidelines in Ham's F-12K (Kaighn's) supplemented with L-glutamine, penicillin/streptomycin (PAA, Cat No: P11-010) and 15% FCS. Reduced concentrations of FCS (5%) were used prior to the assay for 24 h. Composition of KRPG-buffer was (in mM): NaCl (129 mM), KCl (4.86 mM), CaCl2 (1.22 mM), NaH2PO4 (15.8 mM), glucose (5.5 mM), pH 7.3-7.4.

Route of administration:

other: Particles were dispersed according to the NanoGenotox protocol which uses small amounts of serum albumin to stabilize non-polar particles:

Details on exposure:

Cell culture assays were carried out in 96 well plates using 4 concentrations of particles (in triplicates) which were pipetted onto NR8383 cells (3x105 cell/well) under serum-free conditions as previously described1. After 16 h, supernatants were tested for LDH, glucuronidase (GLU) and TNFα-activity (in triplicates). Controls included untreated cells, Triton X-100-treated cells to fully release LDH and glucuronidase, and lipopolysaccharide (LPS)-treated cells to test for the macrophages' TNF production competence. Particle-free controls were run in duplicates for each particle concentration.

Duration of treatment / exposure:

Details are provided in the attached study report.

Details on study design:

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 (dH2O) containing 0.05% bovine serum albumin. Thus, stock suspension contained 2.56 mg particles/mL, 0.5% (vol/vol) ethanol, and 0.05% bovine serum albumin. Stock suspension was ultrasonicated for 16 min. 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 (see below). The resulting suspension was serially diluted in serum-free F12-K medium to obtain concentrations of 180, 90, 45 and 22.5 μg/mL. 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. Overall, this procedure resulted in suspensions which were sufficiently stable for in vitro testing. Nevertheless a partial sedimentation of components occurred such that all suspensions were vortexed prior to use.

LDH activity was tested with Roche Cytotoxicity Detection Kit. Values were background corrected percentages normalized to the Triton X-100-treated control. Untreated cell control amounted to <25 %, which is a typical base-line release of LDH from NR8383 cells under these conditions and does not indicate cell damage.
GLU activity was measured using p-nitrophenyl-β-D-glucuronide as a chromogen. Values give background corrected percentages of the triton-X100 treated control. Cell controls amounted to < 3%, which is a typical base-line release of GLU from NR8383 cells and does not indicate cell damage.
H2O2 concentration was measured in a cumulative manner for 90 min post particle application. Therefore, a parallel approach was carried out in KRPG buffer using the Amplex Red reagent. This assay is internally controlled using a standard concentration of 30 μM H2O2. Further, the cells' competence to produce H2O2 upon addition of zymosan was tested and found to be within the range of historical records.
Rat tumor necrosis factor α was quantified using a specific ELISA provided by bio-techne (Wiesbaden, Germany) according to the manufacturer’s protocol.

Positive control:

Quartz

Details on results:

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 but appeared granular and partly deteriorated.
NR8383 cells exposed to CAS 81-77-6 for 16 h had largely cleared the settled fraction from the bottom of the culture wells, such that dark blue inclusions appeared within cells. A (largely) complete uptake of visible particles occurred up to a concentration of 180 μg/mL.
CAS 81-77-6 elicited minor effects on GLU release of macrophages. The effect was dose dependent and became significant at 90 μg/mL. All other assays were negative.
CAS 81-77-6 is a passive nanomaterial.

Substance/ Particle		LOAEL (µg/mL)				LOAEL x BET 1000 (mm2/mL)					Result
CAS 81- 77-6	BET (m2/g)	LDH	GLU	TNF	ROS	LDH	GLU	TNF	ROS	Assay results underscoring threshold
	36.4	n.s	90	n.s.	n.s	-	3276	-	-	1	passive

n.s.: not significant over the whole concentration range.

For further details it is referred to the attached study report.

Conclusions:

Pigment Blue 60 is passive when taken up by rat alveolar macrophages.

Executive summary:

CAS 81-77-6 up to a concentration of 180 μg/mL was nearly completely taken up by rat alveolar macrophages (NR8383 cells). Macrophage activating effects were small and GLU increased upon 90 μg/mL. Neither cytotoxic (LDH), pro-inflammatory (TNF), nor oxidative effects (H2O2) were observed.

Cellular responses to CAS 81-77-6 were largely similar to those of the negative control corundum.

A particle is classified as to be active if the Low Observed Adverse Effect Concentration (LOAEC) multiplied by the surface area drops below the threshold value of 6000 mm2/mL for at least 2 out of the 4 tests. For CAS 81 -77 -6, only one of the four tests (glucuronidase release) resulted in a value of less than 6000 mm2/ml. Therefore, the substance was found to be passive.

Description of key information

CAS 81-77-6 up to a concentration of 180 μg/mL was nearly completely taken up by rat alveolar macrophages (NR8383 cells). Macrophage activating effects were small and glucuronidase increased upon 90 μg/mL. Neither cytotoxic (LDH), pro-inflammatory (TNF), nor oxidative effects (H2O2) were observed.

Cellular responses to CAS 81-77-6 were largely similar to those of the negative control corundum.

Given the BET value of Blue CAS 81-77-6, and according to the active/passive classification of Wiemann et al. 2016 ( Wiemann et al. Particle Fibre Toxicology 2016, 12:14), the substance was classified as passive.

Quartz D12 served as positive control and was classified as active.

Additional information

Inhalation exposure to particles may cause adverse lung reaction and the activation and/or damage of alveolar macrophage is assumed to be an early key event. The NR8383 rat alveolar macrophage cell line is a well-established in vitro model which has been established to examine the biologic activity of respirable particles (Wiemann et al. J. Nanobiotechnoilogy 12:14). Four parameters are tested in the cell culture supernatant under serum-free conditions, namely H2O2, tumour necrosis factor alpha (TNF), lactate dehydrogenase (LDH), and the lytic enzyme glucuronidase. These effects are relevant for the biological response of alveolar macrophages (AM) and, if occurring also in vivo, for their clearance process of particles in the lung: Release of LDH reflects membrane damage and necrosis of AM. Glucuronidase is a representative of lytic enzymes released from (phago)lysosomes. These enzymes may damage surrounding tissue. H2O2 from AM may lead to oxidative damage of lipids, proteins and/or DNA putting at risk lung cells in the neighbourhood of AM. Finally, TNF is an important pro-inflammatory cytokine with a multitude of effects on various cell types. These include proliferation, apoptosis, and/or triggering further immune responses.

Quartz DQ12 is a well-accepted positive control as it acts progressively inflammatory and fibrotic within the lung. In contrast, corundum particles elicit no such effects in the rat lung even if a lung burden of 5 mg is applied, e.g. by intratracheal instillation. This remarkable difference between both types of mineral particles is reflected by in vitro testing with primary as well as cultured alveolar macrophages (NR8383) under serum-fee conditions. These particles were, therefore, be used for positive and negative control experiments, respectively.

Sedimentation and uptake of particles was documented on micrographs. Particle size distribution was monitored via particle tracking analysis in different relevant media.