Barium bis[2-chloro-5-[(2-hydroxy-1-naphthyl)azo]toluene-4-sulphonate]

Basic toxicokinetics

Administrative data

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

Cross-reference 1

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

specific investigations: other studies

Remarks:

static solubility

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according 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:

OECD draft TG on “solubility in aqueous media”, restricted to the „screening method“, using a physiological simulant medium.

GLP compliance:

no

Type of method:

other: in chemico

Endpoint addressed:

repeated dose toxicity: inhalation

other: static solubility of this nano material

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).

INFORMATION ON NANOMATERIALS
- Chemical Composition: 96.6% w/w purity
- Particle size & distribution: 30.2 nm (D50)
- Specific surface area: 49 m2/g
- Shape of particles: multimodal
- Surface area of particles: no surface treatment
- Coating: uncoated
- Residual solvent: 0.9% water

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.

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.

Conclusions:

The results of the static solubility testing show that the soluble fraction for Pigment Red 53:1 is not significantly above the limit of detection.

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

specific investigations: other studies

Remarks:

dynamic dissolution

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

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:

repeated dose toxicity: inhalation

other: dynamic dissolution of this nano material

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).

INFORMATION ON NANOMATERIALS
- Chemical Composition: 96.6% w/w purity
- Particle size & distribution: 30.2 nm (D50)
- Specific surface area: 49 m2/g
- Shape of particles: multimodal
- Surface area of particles: no surface treatment
- Coating: uncoated
- Residual solvent: 0.9% water

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.

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.

Conclusions:

Pigment Red 53:1 shows a consistent dissolution detected by UV-Vis spectroscopy over the measurement period and is classified as slightly soluble.

Cross-reference 3

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

specific investigations: other studies

Remarks:

Surface reactivity (abiotic)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

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:

repeated dose toxicity: inhalation

other: surface reactivity (abiotic) of this nano material

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).

INFORMATION ON NANOMATERIALS
- Chemical Composition: 96.6% w/w purity
- Particle size & distribution: 30.2 nm (D50)
- Specific surface area: 49 m2/g
- Shape of particles: multimodal
- Surface area of particles: no surface treatment
- Coating: uncoated
- Residual solvent: 0.9% water

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.

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.

Conclusions:

Pigment Red 53:1 is classified as “passive” in the classic FRAS assay. In the complementary EPR assay, significant signals were produced by Pigment Red 53:1.

Cross-reference 4

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

specific investigations: other studies

Remarks:

surface reactivity in vitro

Type of information:

experimental study

Adequacy of study:

key 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

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

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).

INFORMATION ON NANOMATERIALS
- Chemical Composition: 96.6% w/w purity
- Particle size & distribution: 30.2 nm (D50)
- Specific surface area: 49 m2/g
- Shape of particles: multimodal
- Surface area of particles: no surface treatment
- Coating: uncoated
- Residual solvent: 0.9% water

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.

Details on results:

Pigment Red 53:1 was completely taken up by alveolar macrophages (NR8383 cells) up to a concentration of 180 μg/mL. Cytotoxic or activating effects were not observed. The substance also induced no TNFα formation. H2O2 production could not be measured due to strong optical interference. Due to these properties, cell responses to Pigment Red 53:1 were not different from those to the negative control corundum.
Given the BET value of Pigment Red 53:1 and according to the active/passive classification of Wiemann et al 2016, the substance was classified as to be passive.

Conclusions:

Given the BET value of Pigment Red 53:1 and according to the active/passive classification of Wiemann et al 2016, the substance was classified as to be passive.

Materials and methods

Objective of study:

distribution

other: properties of nano particles

Principles of method if other than guideline:

Static solubility and dynamic dissolution
OECD draft TG on “solubility in aqueous media”, restricted to the „screening method“, using a physiological simulant medium.
ISO:TR19057:2017,(Nti 2017), Nanotechnologies — Use and application of acellular in vitro tests and methodologies to assess nanomaterial biodurability
Sample preparation for the measurement of the solubility of pigments, following the method ETAD-229
Surface reactivity (abiotic)
ISO TS 18827:2017 Nanotechnologies — Electron spin resonance (ESR) as a method for measuring reactive oxygen species (ROS) generated by metal oxide nanomaterials.
Note: ESR is a synonym of EPR, and designates the same setup.
The Ferric Reduction Ability of Serum (FRAS) assay is described in:
Hellack, B., C. Nickel, C. Albrecht, T. A. J. Kuhlbusch, S. Boland, A. Baeza-Squiban, W. Wohlleben and R. P. F. Schins (2017). "Analytical methods to assess the oxidative potential of nanoparticles: a review." Environmental Science: Nano 4: 1920-1934.
Hsieh, Shu-Feng, Dhimiter Bello, Daniel F. Schmidt, Anoop K. Pal, Aaron Stella, Jacqueline A. Isaacs, and Eugene J. Rogers. 2013. 'Mapping the Biological Oxidative Damage of Engineered Nanomaterials', Small, 9: 1853-65.
Gandon, Arnaud, Kai Werle, Nicole Neubauer, and Wendel Wohlleben. 2017. 'Surface reactivity measurements as required for grouping and

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Barium bis[2-chloro-5-[(2-hydroxy-1-naphthyl)azo]toluene-4-sulphonate]

EC Number:

225-935-3

EC Name:

Barium bis[2-chloro-5-[(2-hydroxy-1-naphthyl)azo]toluene-4-sulphonate]

Cas Number:

5160-02-1

Molecular formula:

C17H13ClN2O4S.1/2Ba

IUPAC Name:

barium(2+) bis(5-chloro-2-[(1E)-2-(2-hydroxynaphthalen-1-yl)diazen-1-yl]-4-methylbenzene-1-sulfonate)

Test material form:

solid: nanoform

Details on test material:

- Substance type: organic
- Physical state: solid
- Lot/batch no: HW-08-05

- State of aggregation: solid
- Mass-specific surface area (BET): 49 m2/g
- TEM (min. Feret): 30.2 nm (D50)
- Shape of particles: multimodal
- Surface area of particles: no surface treatment
- Coating: uncoated
- Residual solvent: 0.9% water

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.

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).

INFORMATION ON NANOMATERIALS
- Chemical Composition: 96.6% w/w purity
- Particle size & distribution: 30.2 nm (D50)
- Specific surface area: 49 m2/g
- Shape of particles: multimodal
- Surface area of particles: no surface treatment
- Coating: uncoated
- Residual solvent: 0.9% water

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.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on distribution in tissues:

The pigment was shown to be taken up by alveolar macrophages and did not provoke cytotoxic effects or an increase of inflammation markers. After uptake the particles of Pigment Red 53:1 are expected to dissolve slowly in the phagolysosomal fluid. The particles are furthermore of low to no surface reactivity indicating no potential to induce oxidative damage in contact with alvoelar cells and macrophages.

Any other information on results incl. tables

We followed the advice of ProSafe to use the OECD draft technical guidance on “solubility in aqueous media” to examine the static solubility and dynamic dissolution of Pigment Red 53:1. Because organic pigments are handled as powders, 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. We selected the pH 4.5 phagolysosomal simulant fluid as suitable test medium.

The results of the static solubility testing show that the soluble fraction for Pigment Red 53:1 is not significantly above the limit of detection.

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. Pigment Red 53:1 shows a consistent dissolution detected by UV-Vis spectroscopy over the measurement period and is classified as slightly soluble.

The ability to induce biological oxidative damage was analyzed by FRAS (Ferric Reduction Ability of Serum) assay and EPR (Electron Paramagnetic Resonance spectroscopy). Pigment Red 53:1 is classified as “passive” in the classic FRAS assay. In the complementary EPR assay, significant signals were produced by Pigment Red 53:1.

Thus, under the conditions of these assays, Pigment Red 53:1 is considered to be insoluble to very slightly soluble in phagolysosomal simulant fluid and provokes borderline results when tested for surface reactivity tests in chemico. The outcome of the FRAS assay was supported by the surface reactivity assay in vitro: the pigment was completely taken up by alveolar macrophages (NR8383 cells) up to a concentration of 180 μg/mL. Cytotoxic or activating effects were not observed. The substance also induced no TNFα formation. H2O2 production could not be measured due to strong optical interference. Due to these properties, cell responses to Pigment Red 53:1 were not different from those to the negative control corundum. Given the BET value of Pigment Red 53:1 and according to the active/passive classification of Wiemann et al 2016, the substance was classified as to be passive.

As a conclusion, the pigment was shown to be taken up by alveolar macrophages and did not provoke cytotoxic effects or an increase of inflammation markers. After uptake the particles of Pigment Red 53:1 are expected to dissolve slowly in the phagolysosomal fluid. The particles are furthermore of low to no surface reactivity indicating no potential to induce oxidative damage in contact with alvoelar cells and macrophages.

Applicant's summary and conclusion

Conclusions:

As a conclusion, the pigment was shown to be taken up by alveolar macrophages and did not provoke cytotoxic effects or an increase of inflammation markers. After uptake the particles of Pigment Red 53:1 are expected to dissolve slowly in the phagolysosomal fluid. The particles are furthermore of low to no surface reactivity indicating no potential to induce oxidative damage in contact with alvoelar cells and macrophages.