Tetramethyl 2,2'-[1,4-phenylenebis[imino(1-acetyl-2-oxoethane-1,2-diyl)azo]]bisterephthalate

Specific investigations: other studies

Administrative data

Endpoint:

biochemical or cellular interactions

Remarks:

in vitro alveolar macrophage assay

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2020

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Tetramethyl 2,2'-[1,4-phenylenebis[imino(1-acetyl-2-oxoethane-1,2-diyl)azo]]bisterephthalate

EC Number:

271-176-6

EC Name:

Tetramethyl 2,2'-[1,4-phenylenebis[imino(1-acetyl-2-oxoethane-1,2-diyl)azo]]bisterephthalate

Cas Number:

68516-73-4

Molecular formula:

C34H32N6O12

IUPAC Name:

tetramethyl 2,2'-{1,4-phenylenebis[imino(1,3-dioxobutane-2,1-diyl)diazene-2,1-diyl]}diterephthalate

Test material form:

solid: nanoform, no surface treatment

Details on test material:

- State of aggregation:
- Particle size distribution:

- Shape of particles:
- Surface area of particles: BET = 46.3 m2/g
- Crystal structure: crystalline
- Coating: none
- Batch: 13007010
- solid yellow

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: BASF Colours and Effects GmbH

yellow solid
storage at room temperature

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.

Test animals

Species:

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

Administration / exposure

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)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

22.5 other: µg/mL

Doses / concentrations 2

Dose / conc.:

45 other: µg/mL

Doses / concentrations 3

Dose / conc.:

90 other: µg/mL

Doses / concentrations 4

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

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

Results and discussion

Details on results:

Under cell culture conditions, i.e. in F-12K medium, the H2O dispersed material showed a slight increase (approximately 15 % of the mode value) in hydrodynamic diameter (Table 1) indicating some agglomeration. Importantly, there was a layer of micron-sized agglomerates/aggregates at the bottom of the cell culture vials (see Figure ). The size of these aggregates/agglomerates was mostly <5 μm. The density of these settled agglomerates, which are not included in the PTA measurements, correlated with the administered concentration. The majority of the settled aggregates/agglomerates was engulfed by NR8383 cells most of which bore colored inclusions. At the highest concentration many particles were not engulfed at the end of the incubation period and were found among the cells (Figure, bottom right).


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 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 (see Figure, bottom).
Effects on the release of lactate dehydrogenase (LDH), glucuronidase (GLU), H2O2 and TNFα are shown in Table 2. All values and standard deviations (SD) from three independent experiments are summarized in Table 2.
The test material elicited minor effects on the release of LDH at 180 μg/mL. There were no effects on the release of either GLU, TNFα, or H2O2 from NR8383 macrophages.

The gravitationally settled fraction of 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.
There were minor cytotoxic effects at the highest concentration only (LDH). Neither activating (GLU), nor pro-inflammatory effects (TNFα) were observed up to 180 μg/mL.
There was no induction of H2O2 release up to 180 μg/mL.
According to the active/passive classification criteria of Wiemann et al. 2016, the substance was classified as passive.

Any other information on results incl. tables

Table 1: Hydrodynamic diameter (nm) of the test material in H2O, KRPG and F-12K-medium after dispersion according to the NanoGenotox protocol

	mg/L	average	± SEM	average	± SEM	average	± SEM	average	± SEM	average	± SEM
H20	12.8	170	6.3	126.2	4.9	97.5	01. Jan	146.3	4.2	266.1	19
F-12K	6.4	192.1	2.5	141.4	5.2	112.2	1.4	160.7	4.2	311.3	13.5
KRPG	6.4	213.3	5.7	159.3	4.3	127.1	1.9	188.1	10	326	9.8