Benzoic acid, 4-[2-(2-hydroxy-6-sulfo-1-naphthalenyl)diazenyl]-, strontium salt (2:1)

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:

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

Test material information

Constituent 1

Test material form:

solid: nanoform, no surface treatment

Details on test material:

- Batch: PG10631
- Appearance: solid orange
- Shape of particles: see boundary composition
- Surface area of particles: BET 20.3 m2/g
- Crystal structure: crystalline
- Coating: none
- Expiry date: 2022

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Colours and Effects GmbH
-solid, orange
- Purity: >95%

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:

A particle is classified as to be active if the Low Observed Adverse Effect Concentration (LOAEC) 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.
Pigment Orange 79 was active in only one of the four assays (LOEC x BET (mm2/mL) = 3654). Therefore, the overall result of the test battery is "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 (Measured concentrations were 12.8 mg/L)

	Concentration	HD (mean)	HD (mode)	HD (D10)	HD (D50)	HD (D90)
Diluent	Particles/ml	Average ± SEM	Average ± SEM	Average ± SEM	Average ± SEM	Average ± SEM
H20	1.81 x 10xep9	160.2 ± 1	132.2 ± 4.1	114.6 ± 1.8	151.4 ± 3.5	222.2 ± 3.8
KRPG	1.97 x 10exp9	186.3 ± 1.3	155.5 ± 8.7	131.2 ± 1.1	173.5 ± 2.3	258.3 ± 3.2
F-12K	1.70 x 10exp9	212.6 ± 2.7	170.4 ± 7.2	144.7 ± 1.9	203 ± 4.4	299.38 ± 8.4

The H2O-dispersed material showed a slight decrease in hydrodynamic diameter (mode value) upon transfer to KRPG buffer or F-12K medium, of 13 or 52 %, respectively (Table 1), indicating that the dispersion was stable under cell culture conditions. In line with this, cell culture supernatants showed a strong coloration throughout the experiment. In addition, there were a few, non-dispersed micron-sized agglomerates/aggregates (mostly <10 μm) which settled to the bottom of the cell culture vials. The number of these settled agglomerates/aggregates, which are not included in the PTA measurements, correlated with the administered particle concentration (Figure). All settled particulates were ingested by macrophages and were visible in some cells as dark inclusions at the end of the treatment (Figure, lower right).

In vitro toxicity data

NR8383 cells exposed to Pigment Orange 79 (Strontium Salt) had internalized the settled fraction of particles from the bottom of the culture wells up to a concentration of 180 μg/mL Pigment Orange 79 (Strontium Salt) was not cytotoxic and elicited no significant effects on the release of LDH, GLU, and H2O2 from NR8383 macrophages up to a concentration of 180 μg/mL. There was a very low though dose-dependent increase of TNFα which became significant at the highest particle concentration only.

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.

Sterility Testing

The suspension of Pigment Orange 79 (Strontium Salt) did not give any positive results, neither on casein-peptone nor on malt agar during the 72 h incubation period at 37oC. Light microscopic inspection of the diluted suspension at the end of the incubation period gave also no indication for a contamination of test materials with live germs.

Applicant's summary and conclusion

Conclusions:

Pigment Orange 79 (Strontium Salt) was administered to the NR8383 cells as a well dispersed suspension. While the uptake of few larger particles (<10 μm) was obvious from micrographs, the degree of internalization of the small diffusible particle fraction could not be estimated and appeared incomplete. Under these conditions Pigment Orange 79 (Strontium Salt) elicited neither cytotoxic (LDH), activating (GLU) nor oxidative effects (H2O2). A minimal pro-inflammatory effect (TNFα) was noted at the highest concentration only.
Taking into account the BET value of 20.3 m²/g and the active/passive classification criteria of Wiemann et al. 2016m Pigment Orange 79 (Strontium Salt) was classified as passive.

Alveolar macrophages (AMs) play an outstanding role during inhalation exposure since they effectively clear the alveoli from particles. This study addressed the applicability of an in vitro alveolar macrophage assay to distinguish biologically active from passive nanomaterials.