Calcium bis[4-[[1-[[(2-chlorophenyl)amino]carbonyl]-2-oxopropyl]azo]-3-nitrobenzenesulphonate]

Specific investigations: other studies

Administrative data

Endpoint:

specific investigations: other studies

Remarks:

Biodurability and biodissolution in phagolysosomal simulant fluid

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: ISO:TR19057:2017

Version / remarks:

Nti 2017, Nanotechnologies - Use and application of acellular in vitro tests and methodologies to assess nanomaterial biodurability

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD draft TG on “solubility in aqueous media”

Version / remarks:

restricted to the „screening method“

Principles of method if other than guideline:

The solubility of the test item was determined by a static and a dynamic dissolution test after pre-treatment steps for purification of the samples.
Because the test item is handled as powder, the human exposure to dust is considered the most critical scenario, so that dissolution needed to be tested in fluids that are relevant for inhalation. pH 4.5 phagolysosomal simulant fluid (PSF) was selected as recommended by ISO:TR19057:2017 (Nti, 2017, Nanotechnologies. Use and application of acellular in vitro tests and methodologies to assess nanomaterial biodurability).

For the static solubility experiment, the test material was suspended in a pH 4.5 phagolysosomal simulant fluid in a Schott glass bottle and dispersed by continuous ultrasonication during 5 h, followed by shaking to ensure homogeneity for a total incubation of 24 h. After filtration, the dissolved fractions were detected by UV-Vis.

For the dynamic dissolution kinetic experiment, a continuous flow-through system was applied with the test material captured in a flow cell method to determine the biodissolution of materials in relevant lung fluids. The flow cell mimics the non-equilibrium physiological conditions, where ions can be transported away from the lungs. The tempered medium was regulated at a constant flowrate by a pump at 37°C for 7 days and the fluid collection was performed with an autosampler. The particle size was analyzed by UV-Vis.

Furthermore, the dynamic dissolution in gastric simulants and enzymes was analyzed. The sequential-static in vitro setup consists of a stirred beaker with cascaded addition of simulants for saliva, stomach, intestine. The setup and its adaptation to nanomaterials adheres to ISO TS 19057. The chosen media are compliant with the guidance by EFSA (Hardy, A., et al, 2018). For fluids with enzymes, the enzymes were added after the sonication process. The saliva dispersions were stirred for 5 min at a temperature of 37°C. After taking a sample for analysis, the gastric juice was added and adjusted to a pH 2.0. After two hours of stirring at 37°C a second sample was taken, the intestinal fluid was added, and the pH was adjusted to 6.4. Again, after two hours of stirring, the last sample was drawn and analyzed. Immediately after drawing the samples, they were filtered and analyzed by UV-Vis spectroscopy.

GLP compliance:

no

Type of method:

other: in chemico

Endpoint addressed:

basic toxicokinetics

repeated dose toxicity: inhalation

Test material

Test material information

Constituent 1

Reference substance name:

Calcium bis[4-[[1-[[(2-chlorophenyl)amino]carbonyl]-2-oxopropyl]azo]-3-nitrobenzenesulphonate]

EC Number:

276-057-2

EC Name:

Calcium bis[4-[[1-[[(2-chlorophenyl)amino]carbonyl]-2-oxopropyl]azo]-3-nitrobenzenesulphonate]

Cas Number:

71832-85-4

Molecular formula:

C16H13ClN4O7S.1/2Ca

IUPAC Name:

calcium bis[4-({1-[(2-chlorophenyl)carbamoyl]-2-oxopropyl}diazenyl)-3-nitrobenzenesulfonate]

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Batch identification: 21705154
Supplier: Sun Chemical Colors & Effects
CAS No.: 71832-85-4
Purity: 94.5 %
Date of production: 21 Oct 2019
Physical state/appearance: Yellow Powder

Administration / exposure

Route of administration:

other: test material is in direct contact to the phagolysosomal simulant fluid

Vehicle:

other: phagolysosomal simulant fluid (pH 4.5)

Details on exposure:

- For static solubility experiment, the medium contained sodium phosphate dibasic anhydrous (Na2HPO4) 142.0 mg/l; sodium chloride (NaCl) 6650 mg/l; sodium sulfate anhydrous (Na2SO4) 71 mg/l; calcium chloride dihydrate (CaCl2. 2H2O) 29 mg/l; glycine (C2H5NO2) 450 mg/l (as representative of organic acids); potassium hydrogen phthalate (1-(HO2C)–2-(CO2K)–C6H4) 4085 mg/l (as ion scavenger); alkylbenzyl-dimethylammonium chloride (ABDC) 50 ppm (as antifungal agent); NaN3 20 mg/l; conditions at 37 ± 0.5°C.

- For dynamic dissolution kinetic experiment, the medium contained sodium phosphate dibasic anhydrous (Na2HPO4) 142.0 mg/l; sodium chloride (NaCl) 6650 mg/l; sodium sulphate anhydrous (Na2SO4) 71 mg/l; calcium chloride dihydrate (CaCl2.2H2O) 29 mg/l; glycine (C2H5NO2) 450 mg/l (as representative of organic acids); potassium hydrogen phthalate (1-(HO2C)–2-(CO2K)–C6H4) 4085 mg/l (as ion scavenger); alkylbenzyl-dimethylammonium chloride (ABDC) 50 ppm (as antifungal agent). The pH is adjusted by 0.1 m HCl to pH 4.5.

- For dynamic dissolution in gastric simulant and enzymes, the GIT simulant media contained the following components (according to DIN ISO 19738):
Saliva: NaCl (1667 mg/L), NaSCN (500 mg/L), Na2SO4 (1833 mg/L), NaHCO3 (500 mg/L), KCL (1500 mg/L), KH2PO4 (2000 mg/L), CaCl2*2H2O (500 mg/L), Uric acid (333 mg/L), Urea (33 mg/L), not always included: Mucin (2500 mg/L), a-Amylase (833 mg/L)
Stomach: NaCl (4143 mg/L), KCl (1000 mg/L), KH2PO4 (386 mg/L), not always included: Mucin (4286 mg/L), Pepsin (1429 mg/L), conc. HCl (20 µl)
Intestine: KCl (300 mg/L), CaCl2*2H2O (500 mg/L), MgCl2*6H2O (200 mg/L), NaHCO3 (1000 mg/L), Urea (300 mg/L), not always included: Pancreatin (9000 mg/L), Trypsin (300 mg/L), always in intestine: Bile (9000 mg/L)

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

static solubility experiment: 24 hours; dynamic dissolution kinetic experiment: 7 days

Frequency of treatment:

one treatment

Details on study design:

Pre-treatment:
- To avoid false positive results (detection of additives, impurities, etc.) pigments were purified by sequential solvent washes: first methanol/toluol (80/20), then n-octanol, finally methanol.
- For each solvent the pigment was shaken for 2 hours at room temperature, recovered by centrifugal pelletting (30,000 rpm, 3h), and dried under vacuum (1.3 mbar, 90°C, 1h).
- The solvent extracts were analyzed by UV-Vis spectroscopy and discarded.
- Purified pigment samples were used for solubility tests.

Static solubility experiment:
- 1 mg of test substance were suspended in 100 g of the PSF medium in a Schott glass bottle and were dispersed by continuous ultrasonication during 5 h, followed by shaking to ensure homogeneity for a total incubation of 24 h
- The resulting concentration of 10 mg/L was in the range advised for nanomaterial testing by the OECD GD 318 (2020)
- After 24 h, the medium was filtered through a 1 µm glass filter directly followed by a 0.02 µm (= 20 nm) alumina membrane (both of these filter stages are inorganic)
- Thereafter, the filtrates were analyzed by UV-Vis
- Blank controls (no pigment in the dissolution setup) and medium controls (pure medium) were conducted

Dynamic dissolution kinetic experiment:
- Implementaiton of the Continous Flow System (ISO19057:2017), described by Koltermann-Juelly et al. 2018
- Amount of test item: 1 mg solids per flow cell at 2 mL/h fluid flow
- Number of trials: a single flow-cell with up to 14 eluate samplings
- Duration, temperature: the test was performed at 37 ± 0.5 °C for 7 days
- The detection of dissolved fractions provides the ng/cm²/h metric recommended by Oberdörster et al., is grouped in decadic ranges (Koltermann-Jülly et al. 2018). The detection was adapted to organic substances by using UV-Vis spectrometry.

- Oberdörster, G. and T. A. J. Kuhlbusch (2018). "In vivo effects: Methodologies and biokinetics of inhaled nanomaterials." NanoImpact 10(Supplement C): 38-60.
- Koltermann-Jülly, J., J. G. Keller, A. Vennemann, K. Werle, P. Müller, L. Ma-Hock, R. Landsiedel, M. Wiemann and W. Wohlleben (2018). "Abiotic dissolution rates of 24 (nano)forms of 6 substances compared to macrophage-assisted dissolution and in vivo pulmonary clearance: Grouping by biodissolution and transformation." NanoImpact 12: 29-41.

Dynamic Dissolution in Gastric Simulant and Enzymes:
- The sequential-static in vitro setup consists of a stirred beaker with cascaded addition of simulants for saliva, stomach, intestine.
- The setup and its adaptation to nanomaterials adheres to ISO TS 19057. The chosen media are compliant with the guidance by EFSA (Hardy, A., et al, 2018).
- The samples were dispersed with a BRANSON Sonifier 450D at an amplitude of 30% for 16 minutes within an inverted cup-horn sonicator to deliver an energy output of 7.35 W. A 50 mL Nalgene® bottle was used to reduce inorganic impurities. For fluids with enzymes, the enzymes were added after the sonication process.
- The saliva dispersions were stirred for 5 min at a temperature of 37°C. After diverging a sample, gastric juice was added and set to a pH 2.0 to 2.5. During two hours of stirring at 37°C, samples were collected, and finally intestinal fluid was added, and set to a pH of 6.4. Again, during two hours of stirring at 37°C, samples were collected. Immediately after drawing the samples, they were split for analysis of dissolved components and of remaining particles (0.02 µm Al-Si syringe filter). The filtrate was analyzed by UV-Vis spectroscopy (Cary 5000, Agilent Technologies, Santa Clara, CA, US) in either 1-cm-cuvettes with calibration by the extinction coefficient.
- The simulant fluid compositions are standardized in DIN 19738 and currently being harmonized by the OECD project for a new TG “Determination of solubility and dissolution rate of nanomaterials in water and relevant synthetic biologically mediums”.

Examinations

Examinations:

UV-Vis spectrophotometry was used to analyse dissolved fractions that resulted a) from the sample preparation by impurity extraction solvents in methanol/toluene (80:20), n-octanol and methanol, and b) from the filtered physiological fluids during static or dynamic dissolution testing.
The extracted solvents and the filtered physiological fluids were assessed without dilution, to identify extracted impurities.

- The general procedure followed “UV-VIS Absorption Spectra (spectrophotometric method)”, OECD guideline for Testing of Chemicals, guideline 101, adopted 12th May 1981
- Spectrophotometer: Agilent Cary 5000
- Wavelength range: 200-800 nm
- Cell type: quartz, a) 10.0 mm b) 50.0 mm (to optimize detection down to 0.005 absorbance units)
- Calibration of UV-Vis at peak pigment wavelength for quantification of the dissolved material was performed by dissolving the pigment in concentrated sulfuric acid to get the mass attenuation coefficient
- Blank controls (no test item in the dissolution setup) and medium controls (pure medium) were conducted.
- Limit of Detection (LoD): 0.005 absorbance units
- Limit of Quantification (with the specific attenuation coefficient): 0.01 mg/L

Results and discussion

Details on results:

By-products of the test item were removed by extraction with solvents applying the ETAD method 229. The UV-Vis-absorbing impurities between 0.486% and 1.15% were removed from the pigments.

The test item showed a soluble fraction of less than 0.015 mg/L, corresponding to less than 0.15% dissolved fraction and is therefore regarded as insoluble in the static dissolution assay.
Furthermore, the test item showed a dissolution rate of less than 0.039 ng/cm²/h. The test item is also considered as insoluble in the dynamic dissolution assay.
The dynamic dissolution rate determined via ICP-MS was 14.066 ng/cm²/h. The test item was categorized as metal leaching.

After the cascaded incubation in all three GIT, the test item was categorized as borderline to insoluble as they are partially soluble in intestine only. The detected concentrations of the dissolved test material are 0.019 % after saliva, 0.053 % after stomach and 0.179 % after intestine.

Any other information on results incl. tables

Table 2: Results with detection by UV-Vis

	Static dissolution, PSF shaker, dissolved fraction		Dynamic dissolution, Max observed concentration	Dynamic PSF dissolution rate k	Categorization
Test item	<0.015 mg/L	<0.15%	0.013 mg/L	0.003 ng/cm²/h	insoluble
Blank control	< 0.1 mg/L	-
Medium control	< 0.1 mg/L	-

 

Table 3: Results detected by ICP-MS.

	Max observed concentration	Dynamic PSF dissolution rate k	Categorization
Test item	1.400 mg/L	13.618 ng/cm²/h	Metal leaching

 

Table 4: Results of Dynamic Dissolution in Gastric Simulant and Enzyme.

	Dissolved after saliva pH 6.4 [%]	Dissolved after saliva + 2 h stomach pH 2.0 [%]	Dissolved after saliva + 2 h stomach + 2 h intestine pH 7.5 [%]	Categorization
Test item without Enzyme	0.023	0.048	0.206	Partially soluble (in intestine), borderline to insoluble

Applicant's summary and conclusion

Conclusions:

The test item was identified as insoluble in static solubility assay (lung), insoluble but metal leaching in the dynamic dissoultion assay (lung) and partially soluble in the cascaded dissolution assay (GIT).

Overall the test item is identified as partially soluble.

Executive summary:

The static solubility was examined by suspending the test item in phagolysosomal simulant fluid at pH 4.5 for 24h. Thereafter the dissolved fractions were detected by UV-Vis. The test item was insoluble with less than 0.15% dissolved fraction.

The dynamic dissolution assay is an abiotic flow-through method to determine the biodissolution of materials in relevant lung fluids. The flow cell mimics the non-equilibrium physiological conditions, where ions can be transported away from the lungs. A phagolysosomal simulant media with a low pH value (4.5) was used because alveolar macrophages collect and engulf the vast majority of inhaled pulverulent particles from the alveolar surface, and rapidly transfer them into acidic phagolysosomes. The test was performed over seven days, at 37°C. The test item was insoluble but metal leaching with dissolution rates of 0.003 ng/cm²/h (UV-Vis detection) and 13.618 ng/cm²/h (ICP-MS detection).

The cascaded incubation in all three GIT phases was performed with quantification of the dissolved fraction after each GIT phase (0.023 %, 0.048 %, 0.206 %). The test item was partially soluble (in intestine only).