Zeolite, cuboidal, crystalline, synthetic, non-fibrous

Specific investigations: other studies

Administrative data

Endpoint:

specific investigations: other studies

Remarks:

in vitro bioactivity: alveolar macrophage test

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: NR8383 alveolar macrophage test

GLP compliance:

not specified

Remarks:

The assay has successfully been validated against 18 short term-inhalation studies carried out under good laboratory practice (GLP) conditions.

Type of method:

in vitro

Test material

Test material information

Constituent 1

Reference substance name:

Zeolite, cuboidal, crystalline, synthetic, non-fibrous

EC Number:

930-915-9

Cas Number:

1318-02-1

Molecular formula:

M2/nO • Al2O3 • ySiO2 • wH2O (n is the valency of the cation M, predominantly Na, y can range from 0.64 to 8.8, and w is the number of water molecules (general formula) Na: 1.34 - 24.02%, Al: 2.20 - 39.51%, Si: 15.52 - 68.64% (general composition); additionally, depending on the water quality: Ca, Mg and K might be present below 6%

IUPAC Name:

Zeolite, cuboidal, crystalline, synthetic, non-fibrous

Test material form:

solid: particulate/powder

Remarks:

no surface treatment

Specific details on test material used for the study:

Köstrolith, Zeocros CG180, Na Y Zeolite, SYLOSIV A4 (all provided by Grace GmbH)

Examinations

Positive control:

Micron-sized corundum and quartz DQ12 particles were included in the study as negative and positive particle controls, respectively

Results and discussion

Any other information on results incl. tables

Light Microscopic Observations of Particle-Treated Cells

Phase contrast images were taken from cell-free and cell-containing vials at the end of the exposure period. Untreated control cells with smooth surfaces and numerous pseudopodia occurred singly or formed small cell aggregates. In the presence of LPS (used to control for the cells competence to form TNFα) cells formed larger aggregates, which is a typical response of activated cells. Corundum and quartz DQ12 particles were added as negative and positive control materials, respectively, and reacted as expected: Corundum-treated cells were particle-laden but undamaged. Quartz DQ12 treated cells were particle laden and appeared granular and partly deteriorated.

All zeolite particles under study also formed visible precipitates at the bottom of the culture well, whose number and form were highly heterogeneous. Köstrolith, Zeocros CG180, Na Y Zeolite and SYLOSIV A4 were completely ingested by the cells, such that the bottom among cells was free of particles at the end of the treatment period, even at the highest concentration (90 μg/mL). In all these cases cells appeared largely undamaged after 16 h.

 

Particle Concentrations
In the next step the author looked for ultrafine particles present in the stock suspensions. Because nano-sized particles are not detectable by phase contrast microscopy and hardly settle within the incubation period, he analyzed the 16 h supernatant from the particle suspensions with the highest concentration (90 μg/mL).
All materials exhibited comparatively flat curves with multiple peaks ranging from 60 to 500 nm. Mode values as detected by the NTA software ranged from 88.1 to 252.8 nm.

Overall results indicate very low numbers of ultrafine particles in the supernatants of all zeolite suspensions, despite the high concentration of the weighed-in material (90 μg/mL).

 

In vitro Findings
The major aim of the study was to analyze the bioactivity of four zeolite materials using the alveolar macrophage model. Zeolites were administered to NR8383 cells under protein-free conditions at concentrations of 11.25, 22.5, 45, and 90 μg/mL. In the macrophage assay four parameters were tested: three of them, namely lactate dehydrogenase (LDH, a cytoplasmic enzyme), glucuronidase activity (GLU, a (phago)lysosomal enzyme), and tumor necrosis factor α (TNF α) were measured in the cell culture supernatant retrieved from the culture after 16 h. Because H2O2 is not stable and is released from stimulated cells for a limited period only, its concentration was measured in KRPG buffer 90 min post exposure to particles. 

 

Controls

Untreated cell controls were included in all tests, as were particle controls which consisted of micron-sized corundum and quartz DQ12-treated cells (0-180 μg/mL), according to the standard operation procedure. While corundum elicited no adverse responses after 16 h, quartz DQ12 led to cytotoxicity, activation, and pro-inflammatory response indicated by increased release of LDH, GLU, and TNFα, respectively. TNFα responsiveness was further controlled by adding lipopolysaccharide (LPS, 0.5 μg/mL) as a specific inducer. This elicited expectedly high TNFα values (303.5±68.8 pg/mL). The induced formation of H2O2 was measurable upon both, corundum and quartz DQ12 (≤ 2.8 μM), but was expectedly high upon 360 μg/mL zymosan (16.7 ± 0.8 μM), which is a strong inducer of the NADPH oxidase reaction in macrophages. Overall, effects of cell and particle controls were within the borders of historical records, indicating the responsiveness of NR8383 macrophages and the validity of the assay.

 

Tested Aluminosilicates (zeolites)

Köstrolith, Zeocros CG180, Na Y Zeolite and SYLOSIV A4 elicited different responses:

SYLOSIV A4 was the least bioactive material as it was completely taken up by the cell but elicited no responses in all assays.
Köstrolith elicited cytotoxic effects indicated by increased LDH only (≥ 45 μg/mL); effects on other parameters were not observed.
Zeocros CG180 was somewhat more bioactive and moderately increased LDH, GLU, and TNFα, while H2O2 was not produced.
In contrast, Na Y Zeolite led to an increase in H2O2 formation upon 45 μg/mL, but did not influence all other parameters.
Overall, the effects of the four zeolites Köstrolith, Zeocros CG180, Na Y Zeolite, and SYLOSIV A4 on NR8383 macrophages were low or even absent and not uniform.

Applicant's summary and conclusion

Conclusions:

The amount of particles smaller than 200 nm, as measured with particle tracking analysis, was low or hardly measurable. The microscopically visible fractions of all materials were completely ingested by the macrophages. SYLOSIV A4 elicited no effects; Köstrolith induced a release of LDH upon 45 μg/mL (minor cytotoxic effect). Zeocros CG180 moderately increased LDH, GLU, and TNFα, indicating both, cytotoxic and pro-inflammatory effects. Na Y Zeolite increased the H2O2 formation only, a property rarely observed in the macrophage assay.

Executive summary:

Four zeolites, namely Köstrolith, Zeocros CG180, Na Y Zeolite and SYLOSIV A4 were tested for their in vitro bioactivity with the well establish NR8383 alveolar macrophage test. Materials were dispersed in H2O with an ultrasonic dispersion energy of 270 J/mL or 1440 J/mL and administered to the cells under protein-free conditions. The cell culture supernatants were tested for lactate dehydrogenase (LDH), glucuronidase (GLU), and tumor necrosis factor α (TNFα) after 16 h of particle exposure. Hydrogen peroxide (H2O2) production was assessed after 90 min.

Overall, the effects of the four zeolites were heterogeneous and comparatively low when compared to natural aluminosilicates such as bentonite or kaolin studied with the same experimental setup.