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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

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

Constituent 1
Chemical structure
Reference substance name:
N,N'-phenylene-1,4-bis[4-[(2,5-dichlorophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]
EC Number:
223-460-6
EC Name:
N,N'-phenylene-1,4-bis[4-[(2,5-dichlorophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]
Cas Number:
3905-19-9
Molecular formula:
C40H24Cl4N6O4
IUPAC Name:
N,N'-phenylene-1,4-bis[4-[(2,5-dichlorophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Colours and Effects GmbH
- Batch 0004846412
- solid, red
- Purity: ≥ 99 %
- Expiry date: 18 Jan 2023
-storage conditions: ambient (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 / concentrationsopen allclose all
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 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:
While there was no measurable particles fraction upon extensive ultrasonic treatment, the micron-sized, gravitationally settled fraction of the test substance was nearly completely ingested by alveolar macrophages (NR8383 cells) up to a concentration of 180 μg/mL.
There were no cytotoxic (LDH), activating (GLU), or pro-inflammatory effects (TNFα) effects up to the highest concentration. H2O2 concentrations at higher concentrations were not precisely measurable due to optical assay interference.

Under cell culture conditions, i.e. in F-12K medium, the H2O dispersed material showed no increase in hydrodynamic diameter (HD). In KRPG buffer (used for H2O2 measurement) there was an increase in HD of 45% (Table 1), indicating a moderate particle agglomeration. Importantly, there was a homogeneous layer of micron-sized agglomerates/aggregates at the bottom of the cell culture vials, which are not included in the PTA measurements. The density of settled agglomerates correlated with the administered concentration (Figure 1). The size of single aggregates/agglomerates was below 5 μm (Figure 1, upper panels). The major portion of the settled material was engulfed by NR8383 cells (Figure 1). Only at the highest concentration (180 μg/mL), few small particles remained visible among the cells after 16 h.

Taking into account the BET value of 29.5 m²/g and the active/passive classification criteria of Wiemann et al. 2016 the test substance was classified as passive.

Any other information on results incl. tables

Table 1 Hydrodynamic diameters [nm] of test substances in H2O, KRPG, and F-12K medium at 0.018 mg/l.

size (mean) size (mode) size (D10) size (D50) size (D90)
average  ± SEM average  ± SEM average  ± SEM average  ± SEM average  ± SEM
H20 184.5 4 140.8  12.2 94.7  3.4 175.6 4 274.2  6.3
F-12K 173.6  1.8 140.1  14.4 105.4  1.2 163.4  2.3 241.9  4.5
KRPG 189.5  2.1 203.6  14.5 121.1  3.1 183.7  2.9 246.8  6.4

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.

Applicant's summary and conclusion