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Endpoint:
specific investigations: other studies
Remarks:
dynamic dissolution
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
other: ISO:TR19057:2017
Version / remarks:
Nanotechnologies - Use and application of acellular in vitro tests and methodologies to assess nanomaterial biodurability (Nti, 2017)
GLP compliance:
no
Type of method:
in vitro
Endpoint addressed:
other: dynamic dissolution
Specific details on test material used for the study:
- Batch identification: HW-08-05
- Purity: 96.9 %
- Date of production: 2008
- Physical state / appearance: solid / red
- Purification and pre-treatment: To avoid false positivee results (detection of additives, impurities etc.) the substance was purified by sequential solvent washes: first methanol / toluene (80/20, Merck, HPLC grade), then n-octanol (Sigma-Aldrich, Spectrophotometric grade), finally methanol (Merck, HPLC grade). For each solvent the substance is shaken for 2 hours at room temperature, recovered by centrifugal pelleting (20,000 rpm, 1 h), and dried under vacuum (1.3 mbar, 90 °C, 1 h). The solvent extracts are analyzed by UV-Vis spctroscopy and discarded (Measurement of the solubility of different pigments in different "test-solvents", following the method ETAD-229).
Details on results:
Results with detection by UV-Vis
The test substance has a very low but consistent dissolution in the elugram, resulting in a dissolution rate of 1.03 ng/cm²/h, which places this pigment at the borderline of insoluble to very slightly dissolving materials. Due to a technical failure (the reservoir of PSF fluid ran empty), the testing had a duration of 120 h instead of the intended 168 h (= 7 days). The results are thus only indicative for this pigment.
Endpoint:
specific investigations: other studies
Remarks:
static solubility
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
other: OECD draft TG on "solubility in aqueous media"
Version / remarks:
restricted to the "screening method"
GLP compliance:
no
Type of method:
in vitro
Endpoint addressed:
other: static solubility
Specific details on test material used for the study:
- Batch identification: HW-08-05
- Purity: 96.9 %
- Date of production: 2008
- Physical state / appearance: solid / red
- Purification and pre-treatment: To avoid false positivee results (detection of additives, impurities etc.) the substance was purified by sequential solvent washes: first methanol / toluene (80/20, Merck, HPLC grade), then n-octanol (Sigma-Aldrich, Spectrophotometric grade), finally methanol (Merck, HPLC grade). For each solvent the substance is shaken for 2 hours at room temperature, recovered by centrifugal pelleting (20,000 rpm, 1 h), and dried under vacuum (1.3 mbar, 90 °C, 1 h). The solvent extracts are analyzed by UV-Vis spctroscopy and discarded (Measurement of the solubility of different pigments in different "test-solvents", following the method ETAD-229).
Examinations:
The filtrates were analyzed by UV-Vis.
Details on results:
Impurities with detection by UV-Vis
By-products were removed by extraction with solvents applying the ETAD method 229. The UV-Vis absorption spectra matched the pigment spectra, indicating that the solubility of the pigment in solvent masks the extractable by-products. The UV-Vis analysis was not performed on further extraction solvents, but the photographs that document the solvents are essentially colorless solvents and hence indicate low content of by-products and impurities.

Results with detection by UV-Vis
The substance has 2.6 mg/L dissolved fraction, but this still qualifies as "insoluble" according to the SCCS/1606/19 opinion.
Endpoint:
specific investigations: other studies
Remarks:
Surface reactivity (abiotic)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
other: ESR & FRAS
Principles of method if other than guideline:
- ISO TS 18827:2017 Nanotechnologies - Electron spin resonance (ESR) as a method for measuring reactive oxygen species (ROS) generated by metal oxide nanomaterials
- The Ferric Reduction Ability of Serum (FRAS) assay is described in:
Hellack, B. et al. (2017). "Analytical methods to assess the oxidative potential of nanoparticles: a review." Environmental Science: Nano 4: 1920 - 1934.
Hsieh, Shu-Feng et al. (2013). "Mapping the Biological Oxidative Damage of Engineered Nanomaterials", Small, 9: 1853 - 65.
Gandon, Arnaud et al. (2017). "Surface reactivity measurements as required for grouping and read-across: An advanced FRAS protocol", Journal of Physics: Conference Series, 838: 012033.
GLP compliance:
no
Type of method:
in vitro
Endpoint addressed:
other: surface reactivity (abiotic)
Specific details on test material used for the study:
- Batch identification: HW-08-05
- Purity: 96.9 %
- Date of production: 2008
- Physical state / appearance: solid / red
- Purification and pre-treatment: To avoid false positivee results (detection of additives, impurities etc.) the substance was purified by sequential solvent washes: first methanol / toluene (80/20, Merck, HPLC grade), then n-octanol (Sigma-Aldrich, Spectrophotometric grade), finally methanol (Merck, HPLC grade). For each solvent the substance is shaken for 2 hours at room temperature, recovered by centrifugal pelleting (20,000 rpm, 1 h), and dried under vacuum (1.3 mbar, 90 °C, 1 h). The solvent extracts are analyzed by UV-Vis spctroscopy and discarded (Measurement of the solubility of different pigments in different "test-solvents", following the method ETAD-229).
Details on results:
Results on EPR
The EPR assay with DMPO spin trap spans a dynamic range between 1 * 10^12 and 1.5 * 10^13 spins/mL between the negative and positive controls. The biological oxidative damage is given as absolute number of radicals generated by the nanomaterials and trapped by the DMPO.
The measurement principle of EPR is complementary to FRAS, and has the advantage to rely on microwave spectrometry, for which the presence and light absorption of pigments does not interfere. The pigment generates a reactivity signal that is statistically different from the blank control and was also slightly soluble.

Results FRAS
The FRAS assay spans a dynamic range between 2 and nearly 10,000 nmol TEU/m² ENM between the negative and positive controls. The biological oxidative damage is given in trolox-equivalent units (TEU) per surface dose of engineered nanomaterial (ENM).
The pigment generates a negligible oxidative damage that remains on the order of the negative control.
However, it was noticed that the low density of organic pigments (low compared to metal oxide ENM) renders it impossible to completely separate the pigments from human serum after incubation. Some small particles remain suspended in the serum after separation, and their red color interferes with the optical detection of the oxidative damage. It is technically not feasible to further increase the separation efficiency (e.g. by hard centrifugation) without depleting the human antioxidants, which are also organic (biological) colloids. Due to the remaining color, this interference with the colorimetric detection of the FRAS assay may obscure a low reactivity of up to 20 nmol TEU/m² ENM. However, this is not enough to obscure a significant reactivity (above 500 nmol TEU/m² ENM = 10 % of Mn2O3 reactivity, as defined in the ECETOC DF4nanogrouping framework). Thus, the assignment of the diarrylides to "low reactivity" is supported by the FRAS results despite the intereferences. Because interferences were anticipated, the test plan foresees a redundant determination of oxidative damage by a complementary technique, EPR.
Endpoint:
specific investigations: other studies
Remarks:
surface reactivity in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
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:
no guideline followed
Principles of method if other than guideline:
The aim of the study is to describe in vitro effects of rat alveolar macrophages (NR8383) of nanoscaled pigments. Corundum and quartz DQ12 particles served as negative and positive control.
The biologic effects of respirable particles can be determined with an in vitro test using cultured NR8383 rat alveolar macrophages (Wiemann et al., 2016). Four parameters are tested in the cell culture supernatant under serum-free conditions, namely H2O2, tumour necrosis factor alpha (TNFα), lactate dehydrogenase (LDH), and the lytic enzyme glucuronidase. These effects are relevant for the biological response of AM to particles and, if occuring also in vivo, for their clearance process of particles from the lung: Release of LDH reflects membrane damage and necrosis of AM. Glucuronidase is a representative of lytic enzymes released from (phago)lysosomes. These enzymes may damage surrounding tissue. It is released when macrophages are activated or upon destruction of the phagolysosomal membrane. H2O2 from AM may lead to oxidative damage of lipids, proteins and / or DNA putting at risk lung cells in the neighborhood of AM. Finally, TNFα is an important pro-inflammatory cytokine with a multitude of effects on various cell types. These include proliferation, apoptosis and triggering further immune responses. According to Wiemann et al. (2016), particles may be designated as "active particles" of at least 2 criteria are fulfilled at a certain concentration.
In the assay quartz DQ12 is a well accepted positive control as it elicits strong inflammation and acts progressively fibrogenic in the (rat) lung. In contrast, corundum particles alicit no such effects in the rat lung, even if a lung burden of 5 mg is administered, e.g. by intratracheal instillation. Conrundum is therefore used as a negative control. The 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-free conditions.
In case that particles settle completely such that a defined amount of particle mass is internalized by a known number of cells, experiments may provide information on a mean upper particle load tolerated. This may be helpful for planning in vivo experiments. Therefore, sedimentation and uptake of particles are routinely controlled by light microscopy.
GLP compliance:
no
Type of method:
in vitro
Endpoint addressed:
other: surface reactivity in vitro

Description of key information

Concerning static solubility, the soluble fraction of the test substance is significantly above the limit of detection when detected by UV-Vis.

The test substance shows a consistent dissolution over the measurement period and is classified as slightly soluble.

The ability to induce biological oxidative damage was analyzed by FRAS assay and EPR. The pigment is classified as "passive" in the classic FRAS assay. In the complementary EPR assay, significant signals were produced by the tested pigment and it is therefore considered to be slightly soluble and is classified as "active" in the EPR assay.

Additional information

The OECD draft technical guidance "solubility in aqueous media" was used to examine the static solubility and dynamic dissolution of the test material. Because the material is handled as a powder, the human exposure to dust is considered the most critical scenario, so that dissolution needs to be tested in fluids that are relevant for inhalation. The pH 4.5 phagolysosomal simulant fluid was selected as suitable test medium.

Concerning static solubility, the soluble fraction of the test substance is significantly above the limit of detection when detected by UV-Vis.

For the purposes of the dynamic dissolution kinetic study, a "continuous flow system" was implemented. The detection of dissolved fractions provides the ng/cm²/h metric, grouped in decadic ranges. Similar to the conditions of the static solubility testing, dynamic dissolution was performed in phagolysosomal simulant fluid pH 4.5 medium.

The test substance shows a consistent dissolution over the measurement period and is classified as slightly soluble.

The ability to induce biological oxidative damage was analyzed by FRAS assay and EPR. The pigment is classified as "passive" in the classic FRAS assay. In the complementary EPR assay, significant signals were produced by the tested pigment.

The test item is considered to be slightly soluble and is classified as "active" in the EPR assay.