Calcium bis[3-nitro-4-[[2-oxo-1-[(phenylamino)carbonyl]propyl]azo]benzenesulphonate]

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

specific investigations: other studies

Remarks:

Biodurability and biodissolution in phagolysosomal simulant fluid

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2022

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:

according to guideline

Guideline:

other: ISO:TR19057:2017

Version / remarks:

Nti 2017, Nanotechnologies - Use and application of acellular in vitro tests and methodologies to assess nanomaterial biodurability

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD draft TG on “solubility in aqueous media”

Version / remarks:

restricted to the „screening method“

Principles of method if other than guideline:

The solubility of the test item was determined by a static and a dynamic dissolution test after pre-treatment steps for purification of the samples.
Because the test item is handled as powder, the human exposure to dust is considered the most critical scenario, so that dissolution needed to be tested in fluids that are relevant for inhalation. pH 4.5 phagolysosomal simulant fluid (PSF) was selected as recommended by ISO:TR19057:2017 (Nti, 2017, Nanotechnologies. Use and application of acellular in vitro tests and methodologies to assess nanomaterial biodurability).

For the static solubility experiment, the test material was suspended in a pH 4.5 phagolysosomal simulant fluid in a Schott glass bottle and dispersed by continuous ultrasonication during 5 h, followed by shaking to ensure homogeneity for a total incubation of 24 h. After filtration, the dissolved fractions were detected by UV-Vis.

For the dynamic dissolution kinetic experiment, a continuous flow-through system was applied with the test material captured in a flow cell method to determine the biodissolution of materials in relevant lung fluids. The flow cell mimics the non-equilibrium physiological conditions, where ions can be transported away from the lungs. The tempered medium was regulated at a constant flowrate by a pump at 37°C for 7 days and the fluid collection was performed with an autosampler. The particle size was analyzed by UV-Vis.

Furthermore, the dynamic dissolution in gastric simulants and enzymes was analyzed. The sequential-static in vitro setup consists of a stirred beaker with cascaded addition of simulants for saliva, stomach, intestine. The setup and its adaptation to nanomaterials adheres to ISO TS 19057. The chosen media are compliant with the guidance by EFSA (Hardy, A., et al, 2018). For fluids with enzymes, the enzymes were added after the sonication process. The saliva dispersions were stirred for 5 min at a temperature of 37°C. After taking a sample for analysis, the gastric juice was added and adjusted to a pH 2.0. After two hours of stirring at 37°C a second sample was taken, the intestinal fluid was added, and the pH was adjusted to 6.4. Again, after two hours of stirring, the last sample was drawn and analyzed. Immediately after drawing the samples, they were filtered and analyzed by UV-Vis spectroscopy.

GLP compliance:

no

Type of method:

other: in chemico

Endpoint addressed:

basic toxicokinetics

repeated dose toxicity: inhalation

Route of administration:

other: test material is in direct contact to the phagolysosomal simulant fluid

Vehicle:

other: phagolysosomal simulant fluid (pH 4.5)

Details on exposure:

- For static solubility experiment, the medium contained sodium phosphate dibasic anhydrous (Na2HPO4) 142.0 mg/l; sodium chloride (NaCl) 6650 mg/l; sodium sulfate anhydrous (Na2SO4) 71 mg/l; calcium chloride dihydrate (CaCl2. 2H2O) 29 mg/l; glycine (C2H5NO2) 450 mg/l (as representative of organic acids); potassium hydrogen phthalate (1-(HO2C)–2-(CO2K)–C6H4) 4085 mg/l (as ion scavenger); alkylbenzyl-dimethylammonium chloride (ABDC) 50 ppm (as antifungal agent); NaN3 20 mg/l; conditions at 37 ± 0.5°C.

- For dynamic dissolution kinetic experiment, the medium contained sodium phosphate dibasic anhydrous (Na2HPO4) 142.0 mg/l; sodium chloride (NaCl) 6650 mg/l; sodium sulphate anhydrous (Na2SO4) 71 mg/l; calcium chloride dihydrate (CaCl2.2H2O) 29 mg/l; glycine (C2H5NO2) 450 mg/l (as representative of organic acids); potassium hydrogen phthalate (1-(HO2C)–2-(CO2K)–C6H4) 4085 mg/l (as ion scavenger); alkylbenzyl-dimethylammonium chloride (ABDC) 50 ppm (as antifungal agent). The pH is adjusted by 0.1 m HCl to pH 4.5.

- For dynamic dissolution in gastric simulant and enzymes, the GIT simulant media contained the following components (according to DIN ISO 19738):
Saliva: NaCl (1667 mg/L), NaSCN (500 mg/L), Na2SO4 (1833 mg/L), NaHCO3 (500 mg/L), KCL (1500 mg/L), KH2PO4 (2000 mg/L), CaCl2*2H2O (500 mg/L), Uric acid (333 mg/L), Urea (33 mg/L), not always included: Mucin (2500 mg/L), a-Amylase (833 mg/L)
Stomach: NaCl (4143 mg/L), KCl (1000 mg/L), KH2PO4 (386 mg/L), not always included: Mucin (4286 mg/L), Pepsin (1429 mg/L), conc. HCl (20 µl)
Intestine: KCl (300 mg/L), CaCl2*2H2O (500 mg/L), MgCl2*6H2O (200 mg/L), NaHCO3 (1000 mg/L), Urea (300 mg/L), not always included: Pancreatin (9000 mg/L), Trypsin (300 mg/L), always in intestine: Bile (9000 mg/L)

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

static solubility experiment: 24 hours; dynamic dissolution kinetic experiment: 7 days

Frequency of treatment:

one treatment

Details on study design:

Pre-treatment:
- To avoid false positive results (detection of additives, impurities, etc.) pigments were purified by sequential solvent washes: first methanol/toluol (80/20), then n-octanol, finally methanol.
- For each solvent the pigment was shaken for 2 hours at room temperature, recovered by centrifugal pelletting (30,000 rpm, 3h), and dried under vacuum (1.3 mbar, 90°C, 1h).
- The solvent extracts were analyzed by UV-Vis spectroscopy and discarded.
- Purified pigment samples were used for solubility tests.

Static solubility experiment:
- 1 mg of test substance were suspended in 100 g of the PSF medium in a Schott glass bottle and were dispersed by continuous ultrasonication during 5 h, followed by shaking to ensure homogeneity for a total incubation of 24 h
- The resulting concentration of 10 mg/L was in the range advised for nanomaterial testing by the OECD GD 318 (2020)
- After 24 h, the medium was filtered through a 1 µm glass filter directly followed by a 0.02 µm (= 20 nm) alumina membrane (both of these filter stages are inorganic)
- Thereafter, the filtrates were analyzed by UV-Vis
- Blank controls (no pigment in the dissolution setup) and medium controls (pure medium) were conducted

Dynamic dissolution kinetic experiment:
- Implementaiton of the Continous Flow System (ISO19057:2017), described by Koltermann-Juelly et al. 2018
- Amount of test item: 1 mg solids per flow cell at 2 mL/h fluid flow
- Number of trials: a single flow-cell with up to 14 eluate samplings
- Duration, temperature: the test was performed at 37 ± 0.5 °C for 7 days
- The detection of dissolved fractions provides the ng/cm²/h metric recommended by Oberdörster et al., is grouped in decadic ranges (Koltermann-Jülly et al. 2018). The detection was adapted to organic substances by using UV-Vis spectrometry.

- Oberdörster, G. and T. A. J. Kuhlbusch (2018). "In vivo effects: Methodologies and biokinetics of inhaled nanomaterials." NanoImpact 10(Supplement C): 38-60.
- Koltermann-Jülly, J., J. G. Keller, A. Vennemann, K. Werle, P. Müller, L. Ma-Hock, R. Landsiedel, M. Wiemann and W. Wohlleben (2018). "Abiotic dissolution rates of 24 (nano)forms of 6 substances compared to macrophage-assisted dissolution and in vivo pulmonary clearance: Grouping by biodissolution and transformation." NanoImpact 12: 29-41.

Dynamic Dissolution in Gastric Simulant and Enzymes:
- The sequential-static in vitro setup consists of a stirred beaker with cascaded addition of simulants for saliva, stomach, intestine.
- The setup and its adaptation to nanomaterials adheres to ISO TS 19057. The chosen media are compliant with the guidance by EFSA (Hardy, A., et al, 2018).
- The samples were dispersed with a BRANSON Sonifier 450D at an amplitude of 30% for 16 minutes within an inverted cup-horn sonicator to deliver an energy output of 7.35 W. A 50 mL Nalgene® bottle was used to reduce inorganic impurities. For fluids with enzymes, the enzymes were added after the sonication process.
- The saliva dispersions were stirred for 5 min at a temperature of 37°C. After diverging a sample, gastric juice was added and set to a pH 2.0 to 2.5. During two hours of stirring at 37°C, samples were collected, and finally intestinal fluid was added, and set to a pH of 6.4. Again, during two hours of stirring at 37°C, samples were collected. Immediately after drawing the samples, they were split for analysis of dissolved components and of remaining particles (0.02 µm Al-Si syringe filter). The filtrate was analyzed by UV-Vis spectroscopy (Cary 5000, Agilent Technologies, Santa Clara, CA, US) in either 1-cm-cuvettes with calibration by the extinction coefficient.
- The simulant fluid compositions are standardized in DIN 19738 and currently being harmonized by the OECD project for a new TG “Determination of solubility and dissolution rate of nanomaterials in water and relevant synthetic biologically mediums”.

Examinations:

UV-Vis spectrophotometry was used to analyse dissolved fractions that resulted a) from the sample preparation by impurity extraction solvents in methanol/toluene (80:20), n-octanol and methanol, and b) from the filtered physiological fluids during static or dynamic dissolution testing.
The extracted solvents and the filtered physiological fluids were assessed without dilution, to identify extracted impurities.

- The general procedure followed “UV-VIS Absorption Spectra (spectrophotometric method)”, OECD guideline for Testing of Chemicals, guideline 101, adopted 12th May 1981
- Spectrophotometer: Agilent Cary 5000
- Wavelength range: 200-800 nm
- Cell type: quartz, a) 10.0 mm b) 50.0 mm (to optimize detection down to 0.005 absorbance units)
- Calibration of UV-Vis at peak pigment wavelength for quantification of the dissolved material was performed by dissolving the pigment in concentrated sulfuric acid to get the mass attenuation coefficient
- Blank controls (no test item in the dissolution setup) and medium controls (pure medium) were conducted.
- Limit of Detection (LoD): 0.005 absorbance units
- Limit of Quantification (with the specific attenuation coefficient): 0.01 mg/L

Details on results:

By-products of the test item were removed by extraction with solvents applying the ETAD method 229. The UV-Vis-absorbing impurities between 0.486% and 1.15% were removed from the pigments.

The test item showed a soluble fraction of 0.031 mg/L, corresponding to 0.31% dissolved fraction and is therefore regarded as insoluble in the static dissolution assay.
Furthermore, the test item showed a dissolution rate of less than 0.642 ng/cm²/h. The test item is also considered as insoluble in the dynamic dissolution assay.

After the cascaded incubation in all three GIT, the test item was categorized as partially soluble in stomach and intestine. The detected concentrations of the dissolved test material are 0.066 % after saliva, 0.199 % after stomach and 1.362 % after intestine without enzyme.

Table 2: Results with detection by UV-Vis

	Static dissolution, PSF shaker, dissolved fraction		Dynamic dissolution, Max observed concentration	Dynamic PSF dissolution rate k	Categorization
Test item	0.031 mg/L	0.31%	1.169 mg/L	0.642 ng/cm²/h	insoluble (borderline to partially soluble)
Blank control	< 0.1 mg/L	-
Medium control	< 0.1 mg/L	-

 

Table 4: Results of Dynamic Dissolution in Gastric Simulant and Enzyme.

	Dissolved after saliva pH 6.4 [%]	Dissolved after saliva + 2 h stomach pH 2.0 [%]	Dissolved after saliva + 2 h stomach + 2 h intestine pH 7.5 [%]	Categorization
Test item without Enzyme	0.066	0.199	1.362	Partially soluble (in stomach and intestine)
Test item with Enzyme	0.078	0.210	1.013	Partially soluble (in stomach and intestine)

Conclusions:

The test item was identified as insoluble in static solubility assay (lung), insoluble to borderline soluble and metal leaching in the dynamic dissoultion assay (lung) and partially soluble in the cascaded dissolution assay (GIT).

Overall the test item is identified as partially soluble.

Executive summary:

The static solubility was examined by suspending the test item in phagolysosomal simulant fluid at pH 4.5 for 24h. Thereafter the dissolved fractions were detected by UV-Vis. The test item was insoluble with 0.31% dissolved fraction.

The dynamic dissolution assay is an abiotic flow-through method to determine the biodissolution of materials in relevant lung fluids. The flow cell mimics the non-equilibrium physiological conditions, where ions can be transported away from the lungs. A phagolysosomal simulant media with a low pH value (4.5) was used because alveolar macrophages collect and engulf the vast majority of inhaled pulverulent particles from the alveolar surface, and rapidly transfer them into acidic phagolysosomes. The test was performed over seven days, at 37°C. The test item was insoluble to borderline soluble and metal leaching with dissolution rates of 0.642 ng/cm²/h (UV-Vis detection).

The cascaded incubation in all three GIT phases was performed with quantification of the dissolved fraction after each GIT phase (0.066 %, 0.199 %, 1.362 % without enzyme). The test item was partially soluble (in stomach and intestine).

 

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

cell culture study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2020

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 available

Principles of method if other than guideline:

Rat NR8383 alveolar macrophages are exposed as a suspended cell culture to the test material in protein-free cell culture medium. The particle size distribution of the test material in the medium is analyzed by particle tracking in the supernatant. Agglomerates are registered by inspection with a laser illumination system and and inverted microscope with phase contrast optics. Membrane disruption and macrophage activation were assessed by measuring cellular release of lactate dehydrogenase (LDH) and β-glucuronidase (GLU). Besides, pro-inflammatory mediators TNF-α and the induction of oxidative stress was assessed by measuring the formation and release of H2O2.

- Determination of surface reactivity and cytotoxicity in the alveolar macrophage cell line NR8383 is described in Wiemann, M., et al.: An in vitro alveolar macrophage assay for predicting the short-term inhalation toxicity of nanomaterials. J Nanobiotechnology, 2016.14:16.
- Dispersion of the particles is described in the NanoGenotox protocol: Witschger et al.: Final protocol for producing suitable manufactured nanomaterial exposure media, in The generic NANOGENOTOX dispersion protocol –Standard Operation Procedure (SOP) and background documentation. 2011, The National Research Centre for the Working Environment (NRCWE).

GLP compliance:

no

Type of method:

in vitro

Endpoint addressed:

repeated dose toxicity: inhalation

other: cellular surface reactivity on macrophages

Species:

other: NR8383 rat alveolar macrophage cell line

Details on test animals or test system and environmental conditions:

- The NR8383 rat alveolar macrophage cell line was purchased from ATCC and was used and cultured according to ATCC guidelines.
- Culture medium: rat NR8383 cells were cultured in Ham's F-12K supplemented with L-glutamine, penicillin/streptomycin and 15% fetal calf serum (FCS) under standard cell culture conditions (37 °C; 5 % CO2). Reduced concentrations of FCS (5%) were used prior to the assay for 24 h.
- Composition of KRPG-buffer (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: in vitro, test material is in direct contact to rat alveolar macrophage cell line

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Particle suspensions as prepared with the NanoGenoTox protocol were further diluted to measurable concentrations in dH2O, KRPG buffer or F-12K medium (to simulate the conditions of the cell culture) and analyzed by particle tracking analysis, which was carried out with a NanoSight instrument (LM10) equipped with a blue laser (405 nm) and NTA software.

Duration of treatment / exposure:

16 h for measurement of LDH, GLU, and TNF-α release;
1.5 h for measurement of H2O2 formation

Frequency of treatment:

one treatment

Dose / conc.:

180 other: µg/mL

Dose / conc.:

90 other: µg/mL

Dose / conc.:

45 other: µg/mL

Dose / conc.:

22.5 other: µg/mL

Control animals:

yes

Details on study design:

- Liquid suspensions of test material was ultrasonicated and vortexed prior to use.
- Cell culture assays were carried out in 96 well plates using 4 concentrations of particles (in triplicates) which were pipetted onto NR8383 cells (3x10^5 cell/well) under serum-free conditions. After 16 h, supernatants were tested for LDH, 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' TNFα production competence. Particle-free controls were run in duplicates for each particle concentration.

Examinations:

- Cell culture supernatants were harvested and centrifuged for analysis.
- 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 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.
- TNF-α was quantified using a specific ELISA provided by bio-techne according to the manufacturer’s protocol.
- 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.
- Sterility testing: The stock solution of the test substance prepared in F12-K medium were tested for contamination with viable bacteria and/or fungi. Therefore, 50 μl of aqueous stock suspension was plated onto a conventional maltose and a casein peptone agar. Plates were incubated at 37°C for 72h and inspected for colonies of microorganisms after 24, 48 and 72 h.

Positive control:

Quartz DQ12

Details on results:

Sterility Testing:
The suspension of the test substance did not give any positive results, neither on casein-peptone nor on malt agar during the 72 h incubation period at 37 °C. Light microscopic inspection of the diluted suspension at the end of the incubation period also gave no indication for a contamination of test materials with live germs.

Particle Settling and Uptake:
The material was successfully dispersed in distilled H2O (dH2O) following the NanoGenotox protocol. The hydrodynamic diameters (HD) as measured in dH2O and relevant media by PTA are provided in Table 1. Under cell culture conditions, i.e. in F-12K medium, the H2O dispersed material showed an increase (approximately +47.5 % of the mode value) in hydrodynamic diameter (Table 1) indicating some agglomeration. Importantly, there was a layer of uniform micron-sized agglomerates/aggregates at the bottom of the cell culture vials. The size of these aggregates/agglomerates was <5 μm. The density of these settled agglomerates, which are not included in the PTA measurements, correlated with the administered concentration. Of note, the majority of the settled aggregates/agglomerates was engulfed by NR8383 cells most of which bore colored inclusions. At the highest concentration very few particles were not engulfed at the end of the incubation period and were visible among the cells.

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 but appeared granular and partly deteriorated.

NR8383 cells exposed to the test material largely cleared the settled fraction of particles from the bottom of the culture wells up to a concentration of 180 μg/mL, except for some small particle aggregates/agglomerates. However, colored inclusions within the cells indicated a successful uptake of the main particle fraction.
All values and standard deviations (SD) from three independent experiments showing the effects of the test material on the release of lactate dehydrogenase (LDH), glucuronidase (GLU), H2O2 and TNFα are summarized in Table 2.
The test materialelicited minor effects on the release of LDH at 180 μg/mL. However, there were no effects on the release of either GLU, TNFα, or H2O2 from NR8383 macrophages.

Table 1: Hydrodynamic diameters [nm] of test substances in H2O, KRPG, and F-12K medium.

diluent	conc. [µg/mL]	size (mean)			size (mode)			size (D10)			size (D50)			size (D90)
		average		SEM	average		SEM	average		SEM	average		SEM	average		SEM
H2O	12.8	183.9	±	3	145.2	±	14	111.5	±	2.6	174.9	±	3.9	253	±	3.4
F-12K	6.4	198.8	±	5.1	214.2	±	5.1	124.8	±	4.6	196.3	±	3.8	262.1	±	12.1
KRPG	6.4	199.5	±	1.6	187.9	±	22.9	123.4	±	3.5	197.5	±	5.7	261.5	±	3.2

SEM: standard error of the mean; Size values for D10, D50, D90 describe the cumulative particle size distribution at 10%, 50% and 90% of the maximum value. 

 

Table 2: In vitro effects test substance on NR8383 macrophages in comparison to corundum and quartz DQ12 (n=3).

	internal sample number	LDH [% of pos. CTR]			2-way ANOVA	GLU [% of pos. CTR]			2-way ANOVA	ROS H2O2 [µmol/L]			2-way ANOVA	TNF TNFa [pg/mL]			2-way ANOVA
[µg/mL]
Test item	287
0		15.82	±	4.56		1.50	±	0.53		1.75	±	0.61		6.56	±	5.94
22.5		17.51	±	4.67		3.41	±	1.28		1.08	±	0.44		1.38	±	2.39
45		18.90	±	4.81		3.70	±	2.93		0.47	±	0.23		3.41	±	3.05
90		21.15	±	4.86		-5.66	±	6.80		-0.25	±	0.54		5.76	±	6.15
180		22.97	±	5.15		-27.55	±	9.96		-2.02	±	0.50		12.78	±	11.27
corundum	n.d
0		13.98	±	2.86		1.57	±	0.41		1.60654	±	0.1638		6.88	±	5.98
22.5		13.33	±	3.10		1.97	±	0.63		1.56374	±	0.26756		2.05	±	1.81
45		12.77	±	1.87		2.88	±	0.67		1.57498	±	0.19223		1.04	±	0.90
90		13.81	±	2.54		2.21	±	1.12		1.92753	±	0.25669		1.70	±	1.72
180		16.50	±	3.54		2.06	±	0.40		2.03132	±	0.28969		1.46	±	1.29
quartz DQ12	n.d
0		13.98	±	2.86		1.57	±	0.41		1.61	±	0.16		6.88	±	5.98
22.5		14.53	±	4.36		0.36	±	0.05		1.68	±	0.36		0.84	±	0.73
45		14.45	±	1.10		1.15	±	0.70		1.72	±	0.30		2.33	±	2.45
90		31.47	±	8.14	***	4.16	±	1.57		2.10	±	0.51		16.86	±	16.12
180		87.20	±	7.47	***	15.56	±	3.79	***	2.73	±	0.54		86.83	±	32.43	***
zymosan	n.d
360										18.20	±	3.38
LPS	n.d
0.5														1076.64	±	25.54

 Mean values and standard deviations from three independent experiments. LDH: lactate dehydrogenase, GLU: glucuronidase, ROS: reactive oxygen species (H2O2), TNF: tumor necrosis factor α (TNF α ). Values significantly different from cell control are marked by asterisks (***: P ≤ 0.001). Two-way analyses of variance (ANOVA) and Dunnett's test were used to compare means from the control and treated groups.

 

Table 3: Low observed adverse effects concentrations and active/passive calculation.

BET [m^2/g]	LOAEC [µg/mL]				LOAEC x BET 1000 [mm^2/mL]				Assay results underscoring threshold	Result
	LDH	GLU	TNF	ROS	LDH	GLU	TNF	ROS
21.7	90	45	180	n.s.	1909	954	3818	-	3	active

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

Conclusions:

The test substance was classified as active.

Executive summary:

The test material up to a concentration of 180 μg/mL was largely ingested by alveolar macrophages (NR8383 cells), except for small aggregates/agglomerates at the highest concentration.

The test item elicited dose dependent effects on the NR8383cells. The substance was cytotoxic upon 45-90 μg/mL (GLU and LDH), and pro-inflammatory (TNFα) upon 180 μg/mL, while effects on H2O2 were not observed. Given the specific surface (BET value) of the tested sunstance, and according to the active/passive classification criteria of Wiemann et al. 2016, the test substance was classified as active.