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biochemical or cellular interactions
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
study report
Report date:

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:
Type of method:
in vitro
Endpoint addressed:
repeated dose toxicity: inhalation

Test material

Constituent 1
Chemical structure
Reference substance name:
5,5'-(1H-isoindole-1,3(2H)-diylidene)dibarbituric acid
EC Number:
EC Name:
5,5'-(1H-isoindole-1,3(2H)-diylidene)dibarbituric acid
Cas Number:
Molecular formula:
Test material form:
solid: nanoform
Details on test material:
purity: >99%
- Purity test date: (analytical report No.: 08L00151)
- Lot/batch No.: 081012P040
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.

Specific details on test material used for the study:
Pigment Yellow 139
5,5'-(1H-isoindole-1,3(2H)-diylidene)dibarbituric acid
Purity 99.7 %
yellow powder
Batch 200012P040

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

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


Positive control:
Corundum and Quartz DQ12

Results and discussion

Details on results:
Sterility Testing
The suspension of Pigment Yellow 139 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 fine particulate material nearly doubled its hydrodynamic diameter (mode value) from 126.4 nm to
242.5 nm (Table 2), indicating moderate particle agglomeration. Importantly, there was a layer of homogeneous micron-sized agglomerates/aggregates at the bottom of the
cell culture vials (see Figure 4). The size of these aggregates/agglomerates was mostly <5 µm (Figure 4, left column). The density of settled agglomerates, which are not
included in the PTA measurements, correlated with the administered concentration (Figure 4).
The vast majority of the settled aggregates/agglomerates was engulfed by NR8383 cells most of which contained colored inclusions after 16 h of incubation (Figure 4).
Nevertheless, some particles were visible outside the cells and the number of such particles was apparently similar over the whole concentration range (Figure 4, right column).

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 Yellow 139 cleared the vast majority of the settled particle fraction from the bottom of the culture wells up to a concentration of 180 µg/mL (see Figure 4, bottom), except for some particles.Effects of Pigment Yellow 139 on the release of lactate dehydrogenase (LDH), glucuronidase (GLU), H2O2 and TNFα are shown in Figure 5. All values and standard deviations (SD) from three independent experiments are summarized in Table 1.
Pigment Yellow 139 elicited a dose-dependent increase in H2O2 which became significant at concentrations ≥90 µg/mL. There was also a small dose-dependent release of LDH from NR8383 macrophages and values significantly different from control were reached upon ≥180 µg/mL only. No significant changes were found for the release of GLU and TNFα.

Any other information on results incl. tables

Table 1: In vitro effects of Pigment Yellow 139 on NR8383 macrophages in comparison to corundum and quartz DQ12 (n=3).

[μg/mL] LDH
[% of pos.control] 
[% of pos. control] 
Pigment Yellow 139 0 21.4 ± 3.1 1.4 ± 0.2 1.7 ± 0.7 9.2 ± 3.0
22.5 20.2 ± 4.7 0.8 ± 1.1 3.1 ± 1.8 8.4 ± 2.5
45 22.0 ± 4.6 2.1 ± 1.2 4.5 ± 2.5 8.5 ± 3.1
90 23.9 ± 5.3 1.8 ± 0.6 5.8 ± 2.8* 9.1 ± 4.2
180 30.5 ± 3.2* 2.1 ± 0.6 8.5 ± 3.3*** 10.6 ± 4.3
Corundum 0 21.4 ± 3.1 1.4 ± 0.2 1.7 ± 0.7 9.2 ± 3.0
22.5 14.9 ± 1.2 2.4 ± 1.1 1.9 ± 0.8 9.0 ± 3.3
45 19.2 ± 2.6 2.3 ± 0.5 2.0 ± 0.8 9.8 ± 3.6
90 22.4 ± 2.8 3.3 ± 1.0 2.2 ± 0.9 10.6 ± 5.2
180 26.3 ± 0.9 2.4 ± 0.5 2.6 ± 1.0 10.7 ± 4.8
Quartz DQ12 0 21.4 ± 3.1 1.4 ± 0.2 1.7 ± 0.7 9.2 ± 3.0
22.5 16.5 ± 3.1 0.8 ± 0.3 1.9 ± 0.9 9.5 ± 4.4
45 19.3 ± 0.3 1.6 ± 1.1 2.1 ± 0.9 11.2 ± 5.2
90 32.2 ± 4.6** 4.3 ± 1.8* 2.3 ± 1.0 25.8 ± 18.6
  180 91.3 ± 6.0*** 14.5 ± 3.1*** 3.0 ± 1.3 76.7 ± 44.3    ***
Zymosan 360 15.1 ± 2.3
LPS 0.5 349.7 ± 138.9

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.


Table 2: Hydrodynamic diameters [nm] of Pigment Yellow 139 in H2O, KRPG, and F-12K medium

diluent Concentration
mean ± SEM mode ± SEM D10 - mean ± SEM D50 - mean ± SEM D90 - mean ± SEM
H2O 2.90E+09 144.8 ± 0.7 126.4 ± 5.5 104.6 ± 0.6 140.2 ± 1.2 191.5 ± 2
KRPG 6.43E+08 220.2 ± 3.4 227.5 ± 59 103 ± 5 225.1 ± 6.3 323.2 ± 5.8
F-12K 9.79E+08 222.6 ± 5.1 242.5 ± 39 137.1 ± 8.4 225.3 ± 11 305 ± 4.8

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
LDH GLU TNF H2O2 LDH GLU TNF H2O2 Assay results under- scoring
18.4 180 n.s. n.s 90 3312 - - 1656 2 active

Applicant's summary and conclusion

The test material of Pigment Yellow 139 was found to be active in this assay as it triggered both lactate dehydrogenase and hydrogen peroxide release by macrophages in vitro.
Executive summary:

The gravitationally settled fraction of Pigment Yellow 139, up to a concentration of 180 µg/mL, was largely ingested by alveolar macrophages (NR8383 cells). There were few settled but not engulfed particles at all concentration steps. Possibly the agglomeration of ultrafine particles observed with PTA has led to a delayed formation of agglomerates which had not yet been engulfed. There was a dose-dependent increase of H2O2 which became significant upon 90 µg/mL, and a minor though significant release of LDH upon 180 µg/mL. The low cytotoxicity combined with a pronounced induction of H2O2 is an uncommon finding. Possibly, Pigment Yellow 139 causes a (nonspecific) activation/modulation of signaling pathways involved in the generation of reactive oxygen. According to two positive test results, the BET value of 18.4 m2 /g, and the active/passive classification criteria of Wiemann et al. 2016 Pigment Yellow 139 was classified as active.