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Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
it is questionable if centrifugation at 20000xg for 3 x 30 min would be sufficient for phase separation of the nano part

Data source

Reference
Reference Type:
publication
Title:
Dissolution behavior of metal oxide nanomaterials in cell culture medium versus distilled water
Author:
Avramescu, M.L. et al.
Year:
2020
Bibliographic source:
J Nanopart Res (2020) 22:222

Materials and methods

Objective of study:
bioaccessibility (or bioavailability)
Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Series on Testing and Assessment No. 29 (23-Jul-2001): Guidance document on transformation/dissolution of metals and metal compounds in aqueous media
Version / remarks:
test medium, loading and test duration adopted for toxicokinetics assessment
Principles of method if other than guideline:
An internationally agreed guideline does not exist for this test (e.g. OECD). However, similar tests have been conducted with several metal compounds in previous risk assessments (completed under Regulation (EEC) No 793/93) and in recent preparation for Regulation (EC) No 1907/2006.

The test substances were performed on the basis of
- OECD Series on Testing and Assessment no. 318 (2017), Dispersion stability of nanomaterials in simulated environmental media;
- OECD test no. 105 (1995), Water solubility;
- OECD test no. 62 (ENV/JM/MONO(2015)44), Considerations for using dissolution as a function of surface chemistry to evaluate environmental behaviour of nanomaterials in risk assessments. A preliminary case study using silver nanoparticles;
- OECD test no. 86 (ENV/JM/MONO(2018)11), Assessment of biodurability of nanomaterials and their surface ligands; series on the safety ofmanufactured nanomaterials,
- OECD test no. 29 (2002), Guidance document on transformation/dissolution of metals and metal compounds in aqueous media
- OECD test no. 3 (2012), Guidance on sample preparation and dosimetry for the safety testing of manufactured nanomaterials. Series on the safety of manufactured Nanomaterials.

The test media were a cell culture medium and deionized water: lung epithelial cell culture medium (DMEM).
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Titanium dioxide
EC Number:
236-675-5
EC Name:
Titanium dioxide
Cas Number:
13463-67-7
Molecular formula:
O2Ti
IUPAC Name:
dioxotitanium
Test material form:
solid: nanoform
Details on test material:
Nano -TiO2:
- State of aggregation: nano powder (76% anatase, 24% rutile)
- Particle size distribution: 19 nm (anatase), 37 nm (rutile)
- Surface area of particles: 55.55 m²/g

Bulk-TiO2:
- State of aggregation: bulk powder (76% anatase, 24% rutile, anatase and rutile was mixed using the same proportion as in NIST SRM1898)
- Particle size distribution: 1500 nm
- Surface area of particles: ~ 5–8 m²/g
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Nano-TiO2: National Institute of Standards and Technology (Gaithersburg, MD, USA); Bulk-TiO2: US Research Nanomaterials, Inc. (Houston, TX, USA)

INFORMATION ON NANOMATERIALS
- Chemical Composition: TiO2
- Particle size & distribution: 19 nm (anatase), 37 nm (rutile)
- Specific surface area: 55.55 m²/g
Radiolabelling:
no

Test animals

Species:
other: in vitro (lung epithelial cell culture medium)
Details on test animals or test system and environmental conditions:
TEST PRINCIPLE IN BRIEF:
- one cell culture medium
- samples taken after 24 and 48 hours agitation (100 rpm, 1hour/day) at 37 °C
- the study was performed in triplicate
- measurement (ICP-OES) of dissolved titanium concentrations in the top part of supernatant after centrifugation (20000xg, 3 x 30 min) and acidification with HNO3 (to a concentration of 2.5 %).

The aim of this test was to assess the dissolution of TiO2 (nano and bulk) in water and cell culture medium: Dulbecco’s modified Eagle’s medium Nutrient Mixture F-12HAM supplemented with 2% fetal bovine serum (FBS). The test media was selected

Administration / exposure

Duration and frequency of treatment / exposure:
Titanium concentrations in cell culture medium (DMEM) were assessed after 0, 2 and 24 hours of incubation.
Doses / concentrationsopen allclose all
Dose / conc.:
10 other: mg of test item / L respective media
Dose / conc.:
100 other: mg of test item / L respective media
Details on study design:
Test setup
Three replicate flasks per test item (TiO2 nano- or bulk-form) and per test medium (deionized water or DMEM) were prepared with loading of 10 mg/L or 100 mg/L. The required amount of dry ENM powder was pre-wetted in deionized water (1 mL) to form a wet paste and after 20 h was dispersed in deionized water to yield the required concentration. Stock dispersions of metal oxide ENMs (1000 mg/L metal oxide) were prepared in deionized water and sonicated according to the material specific delivered sonication energy (DSE). Freshly prepared stock ENM dispersions (1000 mg/L and 100 mg/L) were diluted 10 times either in deionized water or cell culture media (DMEM).
Samples (50 mL) were incubated in 50-mL polypropylene centrifuge tubes at 37 °C for 24 h in an orbital shaker with 1-h shaking per day at 100 rpm. At each time interval, triplicate samples were withdrawn and aliquots were taken for particle characterization (before separation) by DLS while the remaining dispersions were used to measure the solubilized metal fraction after separation from undissolved particles by ICP-OES. The separation was performed by sequential centrifugation at 20,000×g (3 × 30 min). Between successive centrifugation steps, the samples were carefully transferred by pipette (30 mL first step, 15/20 mL second step) from the top part of supernatant to avoid particle re-suspension. The absence of particles from the resulting supernatant was confirmed by DLS. All final extracts were acidified with HNO3 (to a concentration of 2.5%), and dissolved metal concentrations were measured by ICP-OES. The cell culture medium (DMEM) is buffered at pH range 7.4–7.8 which is maintained after the addition of 2 % FBS.

Reagents:
Deionized water (MilliQ 18.2 MΩ cm)
Nitric acid - high purity (supplied by SEASTAR Chemicals Inc., Sidney, BC, Canada))
Hydrochloric acid - high purity (supplied by SEASTAR Chemicals Inc., Sidney, BC, Canada)

Dulbecco’s modified Eagle’s medium Nutrient Mixture F-12HAM supplemented
with 2% foetal bovine serum(FBS), 45 IU/mL penicillin, and 45 IU/mL streptomycin (Life Technologies Inc., Burlington, ON, Canada): composition of DMEM/F-12, no phenol red (# 21041, Thermo Fisher Scientific Inc., USA):
Glycine
L-Alanine
L-Arginine hydrochloride
L-Asparagine-H2O
L-Aspartic acid
L-Cysteine hydrochloride-H2O
L-Cystine 2HCl
L-Glutamic Acid
L-Glutamine
L-Histidine hydrochloride-H2O
L-Isoleucine
L-Leucine
L-Lysine hydrochloride
L-Methionine
L-Phenylalanine
L-Proline
L-Serine
L-Threonine
L-Tryptophan
L-Tyrosine
Biotin
Choline chloride
D-Calcium pantothenate
Folic Acid
Niacinamide
Pyridoxine hydrochloride
Riboflavin
Thiamine hydrochloride
Vitamin
-Inositol
Calcium Chloride (CaCl2) (anhyd.)
Cupric sulfate (CuSO4-5H2O)
Ferric Nitrate (Fe(NO3)3"9H2O)
Ferric sulfate (FeSO4-7H2O)
Magnesium Chloride (anhydrous)
Magnesium Sulfate (MgSO4) (anhyd.)
Potassium Chloride (KCl)
Sodium Bicarbonate (NaHCO3)
Sodium Chloride (NaCl)
Sodium Phosphate dibasic
(Na2HPO4) anhydrous
Sodium Phosphate monobasic
(NaH2PO4-H2O)
Zinc sulfate (ZnSO4-7H2O)
D-Glucose (Dextrose)
Hypoxanthine Na
Linoleic Acid
Lipoic Acid
Putrescine 2HCl
Sodium Pyruvate
Thymidine

METAL ANALYSIS
- As quality control standards, certified reference materials TM-24.4 and TMDA 64.3 (low- and high-level trace elements in water, respectively) were purchased from Environment Canada (Ottawa, ON) and analysed for dissolved titanium by ICP-OES to determine the accuracy of the applied analytical method.
- External calibration was conducted using matrix-matched standards prepared from 1000 μg/mL Ti high-purity standard solutions (Delta Scientific Ltd., Mississauga, ON, Canada).
- Daily optimization was conducted using ICP-OES wavelength calibration solution containing 5 mg/L Al, As, Ba, Cd, Co, Cr, Cu, Mn, Mo, Ni, Pb, Se, Sr, and Zn and 50 mg/LK in 5% HNO3.
- IZON calibration particles of different diameters (CPC 100 nm, 200 nm) were used for the TRPS

Instrumental and analytical set-up for the ICP-OES instrument:
5100 Synchronous Vertical Dual View (SVDV) Agilent ICP-OES, Agilent Technologies, Canada
Power: 1.2 kW, and
Plasma flow: 12 L/min
Auxiliary flow: 1 L/min
Nebulizer flow: 0.7 L/min
viewing mode: SVDV
wave-length (Ti): 334.941 nm

Instrumental and analytical set-up for the DLS/ELS instrument:
Zetasizer Nano ZSP particle characterization system (Malvern Panalytical, Westborough, MA, USA)
power: 10mW
wave-length: 633 nm
non-invasive backscatter (NIBS) technology detection:
scattering angle: 173°
Laser Doppler electrophoresis in combination with M3-PALS
Details on dosing and sampling:
Loading: Detailed loadings of the test vessels are given in "Any other information on materials and methods incl. tables".

Results and discussion

Main ADME resultsopen allclose all
Type:
other: Dissolution of nano-TiO2 in water, 24 h at 37 °C (10 mg/L loading, centrifugation (20000xg, 3 x 30 min.) for phase separation)
Results:
< LOD Ti (dissolved)
Type:
other: Dissolution of nano-TiO2 in water, 24 h at 37 °C (100 mg/L loading, centrifugation (20000xg, 3 x 30 min.) for phase separation)
Results:
0.0052 mg Ti/L (dissolved)
Type:
other: Dissolution of nano-TiO2 in DMEM, 24 h at 37 °C (10 mg/L loading, centrifugation (20000xg, 3 x 30 min.) for phase separation)
Results:
0.0131 mg Ti/L (dissolved)
Type:
other: Dissolution of nano-TiO2 in DMEM, 24 h at 37 °C (100 mg/L loading, centrifugation (20000xg, 3 x 30 min.) for phase separation)
Results:
0.027 mg Ti/L (dissolved)
Type:
other: Dissolution of bulk-TiO2 in water, 24 h at 37 °C (100 mg/L loading, centrifugation (20000xg, 3 x 30 min.) for phase separation)
Results:
0.00015 mg Ti/L (dissolved)
Type:
other: Dissolution of bulk-TiO2 in DMEM, 24 h at 37 °C (100 mg/L loading, centrifugation (20000xg, 3 x 30 min.) for phase separation)
Results:
0.00029 mg Ti/L (dissolved)

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:
The bioaccessibility of titanium dioxide was determined in vitro by simulating dissolution in the cell culture medium (DMEM), required for culturing lung epithelial cell lines (FE1 cells derived from MutaTMMouse) used for in vitro testing of chemical induced mutagenicity, with a loading of 10 mg/L (nano-TiO2) and 100 mg/L (nano- or bulk-TiO2).

After 24 h in cell culture medium (DMEM, pH 7.87, loading 10 mg/L nano-TiO2) dissolved titanium concentration (operationally defined as the dissolved Titanium fraction after centrifugation (20000xg, 3 x 30 min)) was 0.0131 mg/L (0.17 %). And after 24 h with loading of 100 mg/L nano-TiO2 in cell culture (MDEM, pH 7.77) dissolved titanium concentration was 0.027 mg/L (0.045 %).

After 24 h in deionized water (pH 6.79, loading 10 mg/L nano-TiO2) dissolved titanium concentration (operationally defined as the dissolved titanium fraction after centrifugation (20000xg, 3 x 30 min)) was below limit of detection. And after 24 h with loading of 100 mg/L nano-TiO2 in deionized water (pH 7.69) dissolved titanium concentration was 0.0052 mg/L (0.0083 %).

After 24 h in cell culture medium (DMEM, pH 7.37, loading 100 mg/L bulk-TiO2) dissolved titanium concentration (operationally defined as the dissolved titanium fraction after centrifugation (20000xg, 3 x 30 min)) was 0.00029 mg/L (0.0005 %).
After 24 h in deionized water (pH 6.26, loading 10 mg/L nano-TiO2) dissolved titanium concentration (operationally defined as the dissolved titanium fraction after centrifugation (20000xg, 3 x 30 min)) was 0.00015 mg/L (0.00020 %).

The nano-TiO2 were more soluble than his bulk analogue in both investigated media. The solubility of the nano-form in water was 10x higher than the bulk-form. In cell culture medium, the difference in solubility between nano and bulk was even greater for nano-TiO2 (90× higher than bulk). Overall, the bulk TiO2 showed very low solubility after 24 h in both water (0.0002 %) and cell culture medium (0.0005 %). in this study, the solubility maximum of the metal oxide in water was not reached because the study was terminated after 24 h.
Furthermore, the initial concentration of the nano-TiO2 have varying effects on solubility. The solubility of nano-TiO2 in water was measurable only at 100 mg/L, whereas its solubility at 10 mg/L was below the limit of detection. In DMEM, in contrast, nano-TiO2 solubility was significantly higher (p = 0.009) at 10 mg/L (0.17% for 10 mg/L versus 0.045% for 100 mg/L). But the nano-TiO2 dissolution at either initial concentration may be considered negligible (defined as < 1% by OECD guideline 44 (2015) in both media, even though its solubility was slightly enhanced in DMEM compared with water.

Any other information on results incl. tables

The bioaccessibility of titanium dioxide was determined in vitro by simulating dissolution in the cell culture medium (MDEM), required for culturing lung epithelial cell lines (FE1 cells derived from MutaTMMouse) used for in vitro testing of chemical induced mutagenicity, with a loading of and 10 mg/L (nano-TiO2) and 100 mg/L (nano- or bulk-TiO2). Dissolved concentrations were operationally defined as the dissolved titanium fraction from the top part of supernatant after centrifugation (20000xg, 3 x 30 min), see table 2. After 24 hours, dissolution of nano-TiO2 was highest in cell culture medium (MDEM) at an initial concentration of 10 mg/L.

Table 2: Released amounts of TiO2 ENMs into water and DMEM (metal oxide initial concentration: 10 mg/L and 100 mg/L presented as mean (standard deviation) of triplicates. The results are expressed as mg/L and as percent metal released.

Material

Initial

Time

Dissolved

in   water

 

Dissolved

in   DMEM

 

 

concentration

(h)

mg/L

%

pH

mg/L

%

pH

 

 

 

Mean (SD)

Mean (SD)

Mean (SD)

Mean (SD)

Mean (SD)

Mean (SD)

Nano-TiO2

10 mg/L

0 h

2 h

24 h

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

6.93 (0.21)

6.81 (0.33)

6.79 (0.05)

0.0092 (0.0002)

0.0099 (0.0014)

0.0131 (0.0034)

0.118 (0.002)

0.128 (0.018)

0.170 (0.044)

7.65 (0.04)

7.70 (0.04)

7.87 (0.04)

 

100 mg/L

0 h

2 h

24 h

0.0009 (0.0001)

0.0062 (0.0005)

0.0052 (0.0008)

0.0015 (0.0001)

0.0098 (0.0007)

0.0083 (0.0013)

6.62 (0.10)

6.46 (0.04)

6.69 (0.25)

0.008 (0.0002)

0.046 (0.018)

0.027 (0.008)

0.014 (0.0003)

0.077 (0.029)

0.045 (0.012)

7.06 (0.02)

7.81 (0.02)

7.77 (0.02)

Bulk-TiO2

100 mg/L

0 h

2 h

24 h

n/a

0.00014 (0.00003)

0.00015 (0.00004)

n/a

0.00023 (0.00004)

0.00020 (0.00001)

6.59 (0.07)

6.42 (0.1)

6.26 (0.07)

n/a

0.00037 (0.00009)

0.00029 (0.00018)

n/a

0.0006 (0.0002)

0.0005 (0.0003)

7.46 (0.03)

7.68 (0.04)

7.73 (0.05)

< LOD: below limit of detection

Table 3 shows the influence of particle size on percent solubility of nano and bulk TiO2 after 24-h incubation in deionized water and cell culture medium at an initial metal oxide concentration of 100 mg/L.

Table 3: Influence of particle size (nano versus bulk) on % solubility of TiO2 after 24-h incubation in water and DMEM (Initial TiO2 concentration of 100 mg/L) 

Material

Media

% Dissolved

 

Ratio

pH

 

 

 

Nano

Bulk

Nano/bulk

Nano

Bulk

TiO2

Water

DMEM

0.008 (0.001) %

0.045 (0.012) %

0.0002 (0.00001) %

0.0005 (0.0003) %

42

90

6.69 (0.25)

7.77 (0.02)

6.26 (0.07)

7.73 (0.05)

Results presented as mean (standard deviation) of triplicates.

Method validation summary (ICP-OES)

 

TiO2

Limit of detection (LOD) for titanium: 0.00018 mg/L (in water); 0.00017 mg/L (in DMEM)

 

ICP-OES

The recovery range for TM 24.4 and TMDA 64.3 trace element reference materials was 80–105 % for titanium. Samples were matrix blank corrected.

Cell culture medium (DMEM)

Mean recovery of fortified samples: 88.6–109.7 %

 

Deionized water

Mean recovery of fortified samples: 93.4–104.2 %

Applicant's summary and conclusion

Conclusions:
In both media (MDEM and water) the nano- and bulk-form of titanium dioxide was poorly soluble (< 0.05 %) with solubility depending on the particle size (nano/bulk), the initial concentration and test duration. The solubility of the test item was higher in both media for the nano-form (0.008 % in water; 0.045 % in MDEM). In cell culture medium MDEM, the difference in solubility between nano and bulk was even greater for nano-TiO2 (90× higher than bulk). Overall, the bulk TiO2 showed very low solubility after 24 h in both, water (0.0002 %) and MDEM (0.0005 %). Thus, the solubility was 42x higher for the nano-TiO2 than the bulk-form in water after 24 h. Recovery of total dissolved titanium ranged from 88.6–109.7 % for MDEM and 93.4–104.2 % for water. However, it is questionable if centrifugation at 20000xg for 3 x 30 min would be sufficient for phase separation of the nano part.

In personal consultation with the Fraunhofer IME Institute (Schmallenberg, Deutschland), centrifugation at 200’000 x g for optimal separation of undissolved particles corresponds to their experimental experience. It is therefore assumed that centrifugation at 20’000x g (3 x 30 min) is not sufficient to separate the non-dissolved from the dissolved TiO2 and that partially undissolved TiO2 was also measured.

Cell culture medium MDEM
A maximum of 13.1 μg/L and 27 µg/L total dissolved titanium was measured at an initial concentration of 10 mg/L and 100 mg/L nano-TiO2 in MDEM medium after 24 h. At the initial concentration of 100 mg/L of bulk-TiO2 was a maximum of 0.29 µg/L total dissolved titanium measured in MDEM medium after 24 h.

Deionized water
A maximum of 5.2 µg/L total dissolved titanium was measured at an initial concentration of 100 mg/L nano-TiO2 in water after 24 h. The measured concentrations of dissolved titanium were below the limit of detection for the initial concentration of 10 mg/L of nano-TiO2. At the initial concentration of 100 mg/L bulk-TiO2 was a maximum of 0.15 µg/L total dissolved titanium measured in water after 24 h.
Executive summary:

The objective of this study was to investigate the dissolution of three metal oxide-engineered nanomaterials (ENMS) (CuO, NiO, and TiO2) in water versus cell culture medium.

The test was performed on the basis of OECD Series on Testing and Assessment No. 29 (2001; ENV/JM/ MONO(2001)9),the Standard Operating Procedure for Bioelution Testing of Metals, Inorganic Metal Compounds, and Complex Metal-Containing Materials: Simulated Gastric Fluid (version 09.11.2018 submitted to ECVAM for acceptance) and according to the bioaccessibility test protocol provided by the monitor. The bioaccessibility protocol has been developed on the basis of relevant published methods.

The bioaccessibility protocol has been developed on the basis of relevant published methods.

 

The test medium was selected to simulate the test conditions of an in vitro test which is used for testing chemical-induced mutagenicity.

 

The test item was pre-wetted in deionized water (1 mL) to form a wet paste and after 20 h was dispersed in deionized water to yield the required concentration. Stock dispersions of metal oxide ENMs (1000 mg/L metal oxide) were prepared in deionized water and sonicated according to the material specific delivered sonication energy (DSE). Freshly prepared stock ENM dispersions (1000 mg/L and 100 mg/L) were diluted 10 times either in deionized water or cell culture media (DMEM).

Three replicate flasks per test item (TiO2 nano or bulk-form) and per test medium (deionized water or DMEM) were prepared with loading of 10 mg/L or 100 mg/L.

 

The dissolved test item concentration was specified by measuring the titanium concentration of the test media (deionized water or cell culture medium (DMEM)) under the applied test conditions after the separation of undissolved test item by centrifugation (20000x g, 3 x 30 min). However, it is questionable if centrifugation at 20000xg for 3 x 30 min would be sufficient for phase separation of the nano part.

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