2-cyano-2-[2,3-dihydro-3-(tetrahydro-2,4,6-trioxo-5(2H)-pyrimidinylidene)-1H-isoindol-1-ylidene]-N-methylacetamide

Specific investigations: other studies

Administrative data

Endpoint:

biochemical or cellular interactions

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2022

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:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test:
Rat NR8383 alveolar macrophages are exposed as a suspended cell culture to the test material in protein-free cell culture medium. The particle size distribution of the test material in the medium is analyzed by particle tracking in the supernatent. Agglomerates are registered by inspection with a laser illumination system and and inverted microscope with phase contrast optics. Membrane disruption and macrophage activation were assessed by measuring cellular release of lactate dehydrogenase (LDH) and β-glucuronidase (GLU). Besides, pro-inflammatory mediators TNF-α and the induction of oxidative stress was assessed by measuring the formation and release of H2O2.

- Quartz DQ12 is a well-accepted positive control as it acts progressively inflammatory and fibrotic within the lung. In contrast, corundum particles elicit no such effects in the rat lung even if a lung burden of 5 mg is applied, e.g. by intratracheal instillation. This remarkable difference between both types of mineral particles is reflected by in vitro testing with primary as well as cultured alveolar macrophages (NR8383) under serum-fee conditions. These particles were, therefore, used for positive and negative control experiments, respectively. Most often the response to unknown particle is intermediate to these controls. To test for the capacity of NR8383 cell to produce H2O2 and TNF, zymosan and lipopolysaccharide (LPS) were used as additional positive controls, respectively.

- In case that agglomeration of particles leads to a complete sedimentation followed by an uptake into a defined number of cells, the experiments provide a first information which mean particle load may be tolerated by AM in vivo. Therefore, sedimentation and uptake of particles are routinely controlled and documented on micrographs. For nano-sized materials, which often need a well-adapted preparation of particle suspension according to established protocols, particle size distribution is monitored via particle tracking analysis in different relevant media.

- Determination of surface reactivity and cytotoxicity in the alveolar macrophage cell line NR8383 is described in Wiemann, M., et al., An in vitro alveolar macrophage assay for predicting the short-term inhalation toxicity of nanomaterials. J Nanobiotechnology, 2016. 14: p. 16.
- Dispersion of the particles is described in the NanoGenotox protocol: Witschger et al. Final protocol for producing suitable manufactured nanomaterial exposure media, in The generic NANOGENOTOX dispersion protocol –Standard Operation Procedure (SOP) and background documentation. 2011, The National Research Centre for the Working Environment (NRCWE).

GLP compliance:

no

Type of method:

in vitro

Endpoint addressed:

repeated dose toxicity: inhalation

other: cellular surface reactivity on macrophages

Test material

Test material information

1

Reference substance name:

2-cyano-2-[2,3-dihydro-3-(tetrahydro-2,4,6-trioxo-5(2H)-pyrimidinylidene)-1H-isoindol-1-ylidene]-N-methylacetamide

EC Number:

278-388-8

EC Name:

2-cyano-2-[2,3-dihydro-3-(tetrahydro-2,4,6-trioxo-5(2H)-pyrimidinylidene)-1H-isoindol-1-ylidene]-N-methylacetamide

Cas Number:

76199-85-4

Molecular formula:

C16H11N5O4

IUPAC Name:

2-cyano-N-methyl-2-[3-(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)-2,3-dihydro-1H-isoindol-1-ylidene]acetamide

Test material form:

solid: nanoform

Details on test material:

Yellow powder
Batch 200007P040
Storage conditions: ambient (room temperature)
Expiry date: 01-Feb-2030

- Shape of particles: spherical
- Surface area of particles: 46.6 m2/g
- Crystal structure: crystalline
- Coating: none

Specific details on test material used for the study:

Name of test substance: C.I. Pigment Yellow 185
Purity: 91.7 g/100 g
Batch identification: 200007P040
Date of production: 01.02.2020
Expiry date: 01.02.2030
Homogeneity: given
Storage conditions: room temperature
Water solubility: insoluble
Physical state/appearance: solid yellow
Mass-specific surface area (BET): 46.62 m2/g

Test animals

Species:

other: NR8383 rat alveolar macrophage cell line

Details on test animals or test system and environmental conditions:

- Culture medium: rat NR8383 cells were cultured in Ham's F-12K supplemented with L-glutamine, penicillin/streptomycin and 15% fetal calf serum (FCS) under standard cell culture conditions (37 °C; 5 % CO2). Reduced concentrations of FCS (5%) were used prior to the assay for 24 h.
- Composition of KRPG-buffer (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: test material is in direct contact to rat alveolar macrophage cell line

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.

Duration of treatment / exposure:

16 h for measurement of LDH, GLU, and TNF-α release
1.5 h for measurement of H2O2 formation

Frequency of treatment:

one treatment

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

180 other: µg/mL

Doses / concentrations 2

Dose / conc.:

90 other: µg/mL

Doses / concentrations 3

Dose / conc.:

45 other: µg/mL

Doses / concentrations 4

Dose / conc.:

22.5 other: µg/mL

Control animals:

yes

Details on study design:

- liquid suspensions of test material was ultrasonicated and vortexed prior to use
- cell culture assays were carried out in 96 well plates using 4 concentrations of particles (in triplicates) which were pipetted onto NR8383 cells (3x10^5 cell/well) under serum-free conditions. After 16 h, supernatants were tested for LDH, glucuronidase 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' TNFα production competence. Particle-free controls were run in duplicates for each particle concentration.

Examinations

Examinations:

- Cell culture supernatants were harvested and centrifuged for analysis.
- LDH activity was tested with Roche Cytotoxicity Detection Kit. Values were background corrected percentages normalized to the Triton X-100-treated control. 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 was measured using p-nitrophenyl-β-D-glucuronide as a chromogen. Values give background corrected percentages of the triton-X100 treated control. Cell controls amounted to < 3%, which is a typical base-line release of GLU from NR8383 cells and does not indicate cell damage.
- TNF-α was quantified using a specific ELISA provided by bio-techne according to the manufacturer’s protocol.
- H2O2 concentration was measured in a cumulative manner for 90 min post particle application. Therefore, 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.
- Assay acceptance criteria: Assay results are accepted if quartz DQ12 leads to significant positive results for LDH, GLU and TNF, while all these parameters are not significantly changed by corundum. The tests for H2O2 is accepted if zymosan elevates the H2O2 concentration by more than 10 μmol/L. A small induction of H2O2 (≤3 μmol/L) by the highest concentration of corundum or quartz may occur. This is in accordance with our historical records and does not influence the outcome of the test.
- Sterility testing: The stock solution of the test substance prepared in F12-K medium were tested for contamination with viable bacteria and/or fungi. Therefore, 50 μl of aqueous stock suspension was plated onto a conventional maltose and a casein peptone agar. Plates were incubated at 37°C for 72h and inspected for colonies of microorganisms after 24, 48 and 72 h.

Positive control:

Corundum and Quartz DQ12

Results and discussion

Details on results:

Particle Settling and Uptake:
The material was successfully dispersed in distilled H2O (dH2O) following the NanoGenotox protocol. The hydrodynamic diameter (HD) could be measured in dH2O (Table 1). In dH20 there was a multimodal size distribution of the HD with multiple peaks ranging from 200 – 500 nm. In KRPG buffer and also in F-12K cell culture media particles formed agglomerates such that PTA analysis of diffusible particles was no longer possible. Under cell culture conditions, i.e. in KRPG buffer or F-12K medium, there was a fluffy layer of agglomerates which settled onto the bottom of the cell culture vials; the density of settled agglomerates correlated with the administered concentration. Except for large agglomerates lying on top of the cells all particles and smaller agglomerates were engulfed by NR8383 cells most of which contained colored inclusions after 16 h of incubation.

Sterility Testing:
The suspension of the test material 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.

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 cleared the vast majority of the settled particle fraction from the bottom of the culture wells up to a concentration of 180 μg/mL, except for a few larger agglomerates. Effects of the test material on the release of lactate dehydrogenase (LDH), glucuronidase (GLU), H2O2 and TNFα are summarized in Table 2. The test material elicited a dose-dependent increase in H2O2 and TNFα which became significant at concentrations of ≥45 μg/mL and 180 μg/mL, respectively. There was also a significant release of LDH from NR8383 macrophages at 180 μg/mL. No significant changes were found for the release of GLU up to 45 μg/mL; higher concentrations could not be evaluated due to optical interference of the yellow test material with the blue light (405 nm) used in the colorimetric assay.

Any other information on results incl. tables

Table 1: Hydrodynamic diameter [nm] of the test substance in H2O, KPRG, and F-12K medium.

			Hydrodynamic Diameter [nm]
					D10	D50	D90
	Diluent	concentration [particles/mL]	mean ± SEM	mode ± SEM	mean ± SEM	mean ± SEM	mean ± SEM
Test substance	H2O	8.24E+08	355.9 ± 8.9	307.2 ± 48.8	204.6 ± 12.1	343.7 ± 6.2	513.5 ± 8.1
	KRPG	n.m.	n.m.	n.m.	n.m.	n.m.	n.m.
	F-12K	n.m.	n.m.	n.m.	n.m.	n.m.	n.m.

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. n.m.: not measurable

 

 

Table 2: In vitro effects of the test substance on NR8383 macrophages in comparison to corundum and quartz DQ12 (n=3).

	[µg/mL]	LDH [% of pos. CTR]	GLU [% of pos. CTR]	H2O2 [µmol/L]	TNFα [pg/mL]
Test substance	0	16.1 ± 4.6	1.7 ± 0.4	0.7 ± 0.6	11.2 ± 3.5
	22.5	16.8 ± 3.5	3.2 ± 0.4	1.4 ± 0.4	15.9 ± 1.0
	45	16.8 ± 2.7	3.4 ± 0.7	1.6 ± 0.3 *	15.8 ± 2.2
	90	16.8 ± 2.5	-1.0 ± 2.4	2.2 ± 0.8 ***	25.1 ± 6.8
	180	35.4 ± 11.0 ***	-1.2 ± 1.6	3.7 ± 0.6 ***	47.0 ± 8.7 ***
Corundum	0	16.1 ± 4.6	1.8 ± 0.2	1.2 ± 0.3	11.2 ± 3.5
	22.5	15.5 ± 3.4	2.2 ± 0.2	1.1 ± 0.2	13.5 ± 3.0
	45	18.6 ± 4.5	2.6 ± 0.4	1.3 ± 0.2	13.2 ± 2.5
	90	21.4 ± 4.2	2.9 ± 0.9	1.6 ± 0.2	14.3 ± 2.9
	180	22.4 ± 5.5	3.3 ± 0.2	1.8 ± 0.3	14.8 ± 1.8
Quartz DQ12	0	16.1 ± 4.6	1.8 ± 0.2	1.2 ± 0.3	11.2 ± 3.5
	22.5	15.3 ± 4.4	2.0 ± 1.0	1.2 ± 0.2	12.6 ± 4.7
	45	17.7 ± 5.0	2.7 ± 0.4	1.2 ± 0.2	13.7 ± 4.3
	90	28.8 ± 6.8 *	4.2 ± 0.9 *	1.6 ± 0.0	28.5 ± 10.3 *
	180	69.9 ± 6.7 ***	12.1 ± 1.8 ***	2.2 ± 0.3 **	94.5 ± 24.5 ***
Zymosan	360			14.1 ± 0.4
LPS	0.5				588.4 ± 15.9

  Mean values and standard deviations from three independent experiments. LDH: lactate dehydrogenase, GLU: glucuronidase, ROS: reactive oxygen species (H2O2), TNF: tumor necrosis factor α (TNFα). Values significantly different from cell control are marked by asterisks (*: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001) Two-way analyses of variance (ANOVA) and Dunnett's test were used to compare means from the control and treated groups. Negative values result from color interference in the assay and were not considered for evaluation.

 

 

Table 3: Low observed adverse effect concentrations and active/passive calculation.

Substance/ Particle		LOAEC (µg/mL)				LOAEC x BET (mm^2/mL)					Result
Test substance	BET (m^2/g)	LDH	GLU	TNF	H2O2	LDH	GLU	TNF	H2O2	Assay results under- scoring threshold
	46.62	180	n.e.	180	45	8392	-	8392	2098	1	passive

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. n.s.: not significant over the whole concentration range; n.e.: not evaluable.

Applicant's summary and conclusion

Conclusions:

According to the test substance's BET value only the H2O2 increase occurred below the threshold. Therefore, the test substance might formally be classified as passive. However, a definite statement cannot be made and some bioactivity of the substance cannot be ruled out.

Executive summary:

The gravitationally settled fraction of the test substance, up to a concentration of 180 μg/mL, was ingested by alveolar macrophages (NR8383 cells) except for larger agglomerates remaining on top of the cell layer. Due to this circumstance substance-related effects may by underestimated.
The test substance elicited a dose-dependent release of H2O2 and of TNFα which became significant upon 45 μg/mL and 180 μg/mL, respectively. There was also a significant release of LDH upon 180 μg/mL, indicating some cytotoxicity at high concentration. There were no effects on the release of GLU up to 45 μg/mL but higher concentrations unfortunately could not be evaluated.
According to the test substance's BET value (46.62 m2/g) only the H2O2 increase occurred below the threshold (see Table 3). Therefore, the test substance might formally be classified as passive. However, due to the lack of results for the release of GLU at two relevant concentration steps, and according to the 2-out-of-4 criterion (Wiemann et al. 2016), a definite statement cannot be made. Considering that the test substance elicited increases of at least three parameters while the particle uptake was incomplete, some bioactivity of the substance cannot be ruled out. Overall, the test substance may be classified as borderline.