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specific investigations: other studies
Biodurability and biodissolution in phagolysosomal simulant fluid
Type of information:
experimental study
study currently ongoing
Adequacy of study:
weight of evidence
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 Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
other: ISO:TR19057:2017
Version / remarks:
(Nti 2017), Nanotechnologies - Use and application of acellular in vitro tests and methodologies to assess nanomaterial biodurability
equivalent or similar to 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 organic pigment is determined by a static and a dynamic dissolution test after pre-treatment steps for purification of the samples.
For the static solubility experiment, the test material was suspended in a pH 4.5 phagolysosomal simulant fluid in a Schott glass bottle and shaked automatically for 24 hours after pre-treatment. The sample was analyzed by IR after combustion of its total organic content and specific fuorescence spectra or automated counting by SEM.
For the dynamic dissolution kinetic experiment, a continuous flow system was applied with the test material captured in a flow cell. 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 TEM as the median particle size before and after continuous flow (by manual evaluation of approximately 300 particles).
GLP compliance:
Type of method:
other: in chemico
Endpoint addressed:
basic toxicokinetics
repeated dose toxicity: inhalation

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
Test materials used in this dossier are all considered to fall under the definition of nano-materials according to the European Commission Recommendation 2011/696/EU as the synthesis and manufacturing of this pigment always yields particulate material with a fine particle size distribution.

Administration / exposure

Route of administration:
other: test material is in direct contact to the phagolysosomal simulant fluid
other: phagolysosomal simulant fluid
sodium phosphate dibasic anhydrous (Na2HPO4) 142.0mg/l; sodium chloride (NaCl) 6650 mg/l; sodium sulfate anhydrous (Na2SO4) 71 mg/l; calcium chloride dihydrate (CaCl2. 2H2O) 29 mg/l
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The determination of total organic content is analyzed by combusting the sample completely in a pure oxygen atmosphere on a catalyst in a continuous flow furnace. The formed carbon dioxide is quantified by an IR detector. The total inorganic carbon is measured by adding the sample into a large amount of acid. As a consequence, the carbonates are decomposed to carbon dioxide. The carbon dioxide is purged with a continuous flow of oxygen and quantified by an IR detector.
Duration of treatment / exposure:
- static solubility experiment: 24 hours
- dynamic dissolution kinetic experiment: 7 days
Frequency of treatment:
- static solubility experiment: one time application of medium
- dynamic dissolution kinetic experiment: continuous flow of medium
Post exposure period:
To avoid false positive results (detection of additives, impurities ect.) pigments are purified by sequential solvent washes: first Methanol/Toluol (80/20, Merck, HPLC grade), then N-Octanol (Sigma-Aldrich, Spectrophotometric grade), finally Methanol (Merck, HPLC grade).
For each solvent the pigment is 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 are analyzed by UVVis spectroscopy and discarded.
Details on study design:
static solubility experiment:
- 10 mg of test substance suspended in 80 mL of medium (resulting concentration of 125 mg/L)
- text concentration is an order of magnitude higher than advised for metal oxide nanomaterials in the OECD draft TG on solubility in aquatic media (2017), because the LoD for organics by TOC is higher than for metals by ICPMS (which is the target of the OECD draft TG)
- closed bottle was placed in an IKA shaker to prevent settling
- after incubation, medium is filtered through a 1µm glasfilter directly followed by a 0.02µm (=20nm) aluminum based membrane (inorganic filters)
- after significant organic leaching by the 3kDa filters (as recommended by the OECD draft TG), this set-up was rejected

dynamic dissolution kinetic experiment:
- implementaiton of the Continous Flow Systm (ISO19057:2017), described by Koltermann-Juelly et al 2018
- 1 mg solids per flow cell applied in a single flow-cell
- up to 14 eluate samplings were taken

Results and discussion

Applicant's summary and conclusion