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Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Reference
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:
2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
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
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:
- Principle of test:
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).

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.
GLP compliance:
no
Type of method:
other: in chemico
Endpoint addressed:
basic toxicokinetics
repeated dose toxicity: inhalation
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 sulphate anhydrous (Na2SO4) 71 mg/L; calcium chloride dihydrate (CaCl2. 2H2O) 29 mg/L. The pH was adjusted by addition of 0.1 m HCl to pH 4.5.

- 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 was adjusted by addition of 0.1 m HCl to pH 4.5.
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:
static solubility experiment: 24 hours; dynamic dissolution kinetic experiment: 7 days
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 (20,000rpm, 1h), and dried under vacuum (100 torr, 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 provided the ng/cm²/h metric recommended by Oberdörster et al. (Oberdörster and Kuhlbusch 2018), was grouped in decadic ranges (Koltermann-Jülly, Keller et al. 2018). The detection was adapted to organic substances by using UV-Vis spectrometry. Expecting low dissolution, the initial mass was increased to 10 mg in order to increase the limit of detection.
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
b) from the filtered physiological fluids during static or dynamic dissolution testing
- The extracted solvents and 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: 0.005 absorbance units
- Limit of Quantification (with the specific attenuation coefficient): 0.04 mg/L
Details on results:
By-products of the test item were removed by extraction with solvents applying the ETAD method 229. The UV-Vis absorption spectra of the extractables did not match the absorption of the test item, indicating that impurities that were chemically not identical to the test item, were successfully removed.
The test item did not dissolve above the limit of detection of the method and is therefore regarded as insoluble in the static dissolution assay.
Furthermore, the test item showed a dissolution rate of 0.002 ng/cm²/h, which was far below the threshold of 0.1 ng/cm²/h. Therefore the test item is also considered as insoluble in the dynamic dissolution assay.

Table 1: 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



<0.02 mg/L



<0.2%



0.004 mg/L



0.002 ng/cm²/h



insoluble



Blank control



< 0.1



-



 



 



 



Medium control



< 0.1



-



 



 



 


Conclusions:
The test item was identified as insoluble.
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 a dissolved fraction below the limit of detection.


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 with a dissolution rate far below the threshold of 0.1 ng/cm²/h.

Data source

Materials and methods

Results and discussion

Applicant's summary and conclusion