N,N'-(2,5-dichloro-1,4-phenylene)bis[4-[(2,5-dichlorophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]

Specific investigations: other studies

Administrative data

Endpoint:

biochemical or cellular interactions

Type of information:

experimental study

Adequacy of study:

key 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:

2020

Report date:

2020

Materials and methods

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.

GLP compliance:

no

Type of method:

in vitro

Endpoint addressed:

repeated dose toxicity: inhalation

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-(2,5-dichloro-1,4-phenylene)bis[4-[(2,5-dichlorophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]

EC Number:

255-005-2

EC Name:

N,N'-(2,5-dichloro-1,4-phenylene)bis[4-[(2,5-dichlorophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]

Cas Number:

40618-31-3

Molecular formula:

C40H22Cl6N6O4

IUPAC Name:

N,N'-(2,5-dichloro-1,4-phenylene)bis{4-[(2,5-dichlorophenyl)diazenyl]-3-hydroxy-2-naphthamide}

Test material form:

solid: particulate/powder

Details on test material:

Name: Cromophtal Red K 3900
Batch: 000096B1
CAS 40618-31-3

Water content <0.1% w/w
3-hydroxy-2-napthtoic acid <0.1% w/w
Date of production 12 Mar 2001
solid, red
storage at room temperature

Specific details on test material used for the study:

Purity: approx. 99 %
Date of production: 12 Mar 2001
Expiry date: 08 Aug 2021
Storage conditions: room temperature
Physical state/ appearance: solid red
Water solubility: insoluble
Mass-specific surface area (BET): 41.4 m2/g


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. 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.
Overall, this procedure resulted in a suspension which was sufficiently stable for in vitro testing. Nevertheless a partial sedimentation of components occurred such that all suspensions were vortexed prior to use.

Test animals

Species:

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

Details on test animals or test system and environmental conditions:

For all experiments the alveolar macrophage cell line NR8383 (ATCC) was used and cultured according to ATCC guidelines in Ham's F-12K (Kaighn's) supplemented with L-glutamine, penicillin/streptomycin (PAA, Cat No: P11-010) and 15% FCS. Reduced concentrations of FCS (5%) were used prior to the assay for 24 h. Composition of KRPG-buffer was (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.

Administration / exposure

Route of administration:

other: in cell culture

Vehicle:

other: F-12K medium and KRPG buffer (depending on the respective investigation)

Details on exposure:

The cell culture assays were carried out in 96 well plates using 4 concentrations of particles (in triplicates) which were pipetted onto NR8383 cells (3x105 cell/well) under serum-free conditions. After 16 h, supernatants were tested for LDH, glucuronidase (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' TNF production competence. Particle-free controls were run in duplicates for each particle concentration.
LDH activity was tested with Roche Cytotoxicity Detection Kit. The obtained values were background corrected and expressed as percentages of the Triton X-100-treated control. The 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 activity was measured using p-nitrophenyl-β-D-glucuronide as a chromogen. The obtained values were background corrected and expressed as percentages of the Triton X-100-treated control. The cell controls amounted to < 3%, which is a typical base-line release of GLU from NR8383 cells and does not indicate cell damage.
Hydrogen peroxide concentration was measured cumulatively for 90 min post particle application. 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.
Rat tumor necrosis factor α was quantified using a specific ELISA provided by bio-techne (Wiesbaden, Germany) according to the manufacturer’s protocol.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The particle suspension as prepared with the NanoGenoTox protocol was 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 (Version 3.1).

Duration of treatment / exposure:

16 h (for the determination of LDH, GLU, and TNF-α release)

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 h. For the determination of LDH, GLU, and TNF-α release, cell culture supernatants were sampled after 16 h of incubation.

Examinations

Positive control:

Corundum and Quartz DQ12

Results and discussion

Details on results:

Sterility Testing
The suspension of Pigment Red 214 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.

Under cell culture conditions, i.e. in F-12K medium, the H2O dispersed material showed a decrease (approximately -14 % of the mode value) in hydrodynamic diameter (Table 1) indicating a slight de-agglomeration. Importantly, there was a layer of uniform 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 (Figure).Of note, the settled aggregates/agglomerates were nearly completely engulfed by NR8383 cells most of which bore colored inclusions.

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 Pigment Red 214 nearly completely cleared the settled fraction of particles from the bottom of the culture wells up to a concentration of 180 μg/mL.
Effects of Pigment Red 214 on the release of lactate dehydrogenase (LDH), glucuronidase (GLU), H2O2 and TNFα are shown in Table 2.
The test material elicited no effects on the release of LDH, GLU, TNFα, or H2O2 from NR8383 macrophages up to a concentration of 180 μg/mL.

Any other information on results incl. tables

Table 1: Hydrodynamic diameters [nm] of Pigment Red 214 in H2O, KRPG, and F-12K medium

diluent	conc. [μg/mL]	size (mean) average		SEM	size (mode) average		SEM	size (D10) average		SEM	size (D50) average		SEM	size (D100) average		SEM
H2O	12.8	201	±	4.8	240.5	±	33.5	102.3	±	3.2	195.1	±	6.9	288.7	±	8.7
F-12K	6.4	172	±	3	132.5	±	11.5	97.8	±	3.2	158.3	±	4.8	244.4	±	3.8
KRPG	6.4	176.1	±	7.1	140.1	±	1.8	106.8	±	6.1	160.1	±	5.9	253.1	±	12.1

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 3 Summary

		LOAEC (µg/mL)			LOAEC x BET (mm2/mL)					Result
BET (m2/g)	LDH	GLU	TNF	H2O2	LDH	GLU	TNF	H2O2	Assay results under- scoring threshold
41.4	n.s.	n.s.	n.s	n.s.	-.	-	-	-	0	passive

Applicant's summary and conclusion

Conclusions:

The test material of Pigment Red 214 was found to be passive in this assay.

Executive summary:

The gravitationally settled fraction of Pigment Red 214 up to a concentration of 180 µg/mL was nearly completely ingested by alveolar macrophages (NR8383 cells). There were no cytotoxic (LDH), activating (GLU), or pro-inflammatory effects (TNFα) effects up to the highest concentration. There was no induction of H2O2 release up to 180 µg/mL. Due to the lack of any adverse effect on alveolar macrophages, and according to the active/passive classification criteria of Wiemann et al. 2016, the substance is considered to be passive in regard to particle-induced cytoxicity to macrophages.