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Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Remarks:
Bioaccessibility - transformation/dissolution in artificial physiological media
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Objective of study:
other: Bioaccessibility
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
Deviations:
yes
Remarks:
Bioaccessibility testing: loading of 100 mg/L; five artificial physiological media agitated at 100 rpm, at 37 °C ± 2 °C; sampling after 2h and 24h; determination of Pr, Si and Zr concentrations after filtration by AAS-GF and ICP-OES
Principles of method if other than guideline:
Solubility of test item in simulated human fluids. Principle of test is similar to Transformation/Dissolution testing according to OECD Series 29 (2001)
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Zirconium praseodymium yellow zircon
EC Number:
269-075-7
EC Name:
Zirconium praseodymium yellow zircon
Cas Number:
68187-15-5
Molecular formula:
Pr(x)Zr(1-x)SiO4 0,03≤x≤0,09
IUPAC Name:
Zirconium praseodymium yellow zircon
Test material form:
solid: particulate/powder
Details on test material:
- Name: Colorante amarillo
- EC Name: Zirconium Praseodymium Yellow Zircon
- Substance type: inorganic pigment
- Physical state: solid, yellow powder, odourless
- Storage condition of test material: at room temperature

Test animals

Species:
other: in vitro (simulated human body fluids)

Administration / exposure

Details on exposure:
The test item was exposed to five different test media at a pH range from 1.7 to 7.4. The following synthetic biological fluids were used:
• Gamble’s solution (GMB, pH 7.4) which mimics the interstitial fluid within the deep lung under normal health conditions (de Meringo et. Al. 1994).
• Phosphate-buffered saline (PBS, pH 7.2), which is a standard physiological solution that mimics the ionic strength of human blood serum. It is widely used in the research (e.g. Norlin et al, 2002) and medical health care community (e.g. Hanawa et al, 2004, Okazaki and Gotoh, 2005) as a reference test solution for comparison of data under simulated physiological conditions.
• Artificial sweat (ASW, pH 6.5) which simulates the hypoosmolar fluid, linked to hyponatraemia (loss of Na+ from blood), which is excreted from the body upon sweating. The fluid is recommended in the available standard for testing of nickel release from nickel containing products (EN 1811, 1998).
• Artificial lysosomal fluid (ALF, pH 4.5), which simulates intracellular conditions in lung cells occurring in conjunction with phagocytosis and represents relatively harsh conditions (Moss 1979).
• Artificial gastric fluid (GST, pH 1.7), which mimics the very harsh digestion milieu of high acidity in the stomach (Hamel et al, 1998; ASTM, 2003).

The test media were selected in order to simulate human exposure as far as possible, e.g. skin contact. Ingestion to the gastro-intestinal tract can either be direct, or previously inhaled particles can be translocated from the respiratory tract to the gastro-intestinal tract by mucociliary clearance. It should be stressed though, that the different test media only simulate physiological conditions to a limited extent, as the complexity and function of the real body fluids are difficult to simulate. However, in vitro results in such synthetic biological media can, in a simple way, provide information that could be relevant for a real situation.

The test solutions were prepared using ultra-pure water and chemicals of analytical grades.

The pH of ALF and GMB was adjusted using 50 % NaOH and 25 % HCl, respectively. The pH of ASW and PBS was adjusted with 1% ammonia solution and 50 % NaOH, respectively.

Artificial gastric fluid, pH 1.6, was prepared according to the ASTM standard using 4 g of 25 % HCl solution diluted with ultra -pure water to 1 L (ASTM D5517, 2003).
Duration and frequency of treatment / exposure:
Samples were taken after 2 h and 24h.
Doses / concentrations
Dose / conc.:
100 other: mg of the test item /L artificial media
Details on study design:
Experimental Procedure
Triplicate samples were prepared for exposure in different test media, each for two different time periods. In addition, one blank sample (without addition of test item) containing only the test solution was incubated together with the triplicate samples for each time period. 5 ± 0.5 mg of the test item was weighed using a Mettler AT20 balance with readability of 2 μg, and placed in a PMP Nalgene® jar. 50 mL of the test solution (no adjustment of solution volume to powder mass was made) was then added to the Nalgene® jar containing the test item, before incubated in a Platform- Rocker incubator SI 80 regulated at 37 ± 2 °C. The solution was gently shaken (bi-linearly) with an intensity of 25 cycles per minute for 2 and 24 hours, respectively.
Details on dosing and sampling:
A “standard loading” of 0.1 g/L was selected, which has some physiological relevance. It further allows a comparison of the generated data with results from the OECD Transformation/Dissolution test (OECD, 2001) and similar bioaccessibility tests conducted with other materials under the same conditions .

The time periods for exposure of the test item were selected to have some relevance to the inhalation/ingestion scenario and to enable comparison with other reported metal release/dissolution data generated for similar time periods. The approximate time for the gastric phase of digestion is about 2 hours, and therefore this exposure time period was considered relevant for testing in artificial gastric fluid (Hamel et al, 1998). The
24 hour exposure was selected as a standard time duration that is relatively easy to compare with existing metal release/dissolution data as well as toxicity data for further evaluation of the bioaccessibility of released metals. Moreover, it can be assumed that human exposure to particles last no longer than 24 hours at ambient conditions.

After exposure, the samples were allowed to cool to ambient room temperature before the final pH of the test solution was measured. The test medium was then separated from the powder particles by centrifugation at 10000 rpm for 10 minutes, resulting in a visually clear supernatant with remaining particles in the bottom of the centrifuging tube. Dynamic light scattering, (Malvern Zetasizer nano ZS instrument) was used to confirm the successful removal of all pigment particles. The supernatant solution was decanted into a polypropylene storage flask and acidified to a pH less than 2 (not needed in the case of artificial gastric fluid) with 65 % pure HNO3 prior to solution analysis

Results and discussion

Main ADME results
Type:
other: Bioaccessibility
Results:
Highest dissolution at a loading of 0.1 g/L after 24 hours: Zr: 2.4 ± 0.8 µg/L (ALF), Si: 62.7 ± 5.8 µg/L (GST), Pr: 574 ± 18 µg/L (GST); after 2 hours: Zr: 1.5 µg/L (ASW), Si: 36.7 ± 3.0 µg/L (ALF), Pr: 348 ± 123 µg/L (GST)

Any other information on results incl. tables

BET-analysis:

The specific surface area, measured by BET-analysis is 0.52 m²/g. It should be underlined that this specific surface area is measured by nitrogen absorption and includes also the surface of surface pores.

Abbreviations:

GMB: Gamble´s solution, PBS: phosphate buffered saline, ASW: artificial sweat, ALF: artificial lysosomal fluid, GST: gastric fluid.

Average total concentration of released elements [μg/L] and the standard deviation of triplicate samples in the different media. Blank values for each individual media and exposure period have been subtracted.

(test item) Exposure GMB PBS ASW ALF GST
 Material   period pH 7.4 pH 7.2 pH 6.5 pH 4.5 pH 1.7
ZrSiPr 2 h < LOD < LOD 1.5 0.9 < LOD
Zr release 24 h < LOD < LOD < LOD 2.4 ± 0.8 2.0
ZrSiPr 2 h < blank 25.8 ± 7.4 < blank 36.7 ± 3.0 26.8 ± 10.9
Si release 24 h < blank 6.3 ± 30.2 9.0 ± 3.0 3.8 ± 1.9 62.7 ± 5.8
ZrSiPr 2 h < LOD 12.6 140 ± 118 17.3 348 ± 123
 Pr release 24 h < LOD < LOD 141 ± 96 57.4 ± 70.2 574 ± 18

Release rate of elements given by the BET surface area [μg/cm² h].

(test item) Exposure GMB PBS ASW ALF GST
Material  period pH 7.4 pH 7.2 pH 6.5 pH 4.5 pH 1.7
ZrSiPr 2 h < LOD < LOD 0.0014 0.0008 < LOD
Zr release 24 h < LOD < LOD < LOD 0.0002 ± 0.0001 0.0002
ZrSiPr 2 h < blank 0.026 ± 0.0075 < blank 0.034 ± 0.0036 0.025 ± 0.010
Si release 24 h < blank 0.0005 ± 0.0025 0.0008 ± 0.0003 0.0003 ± 0.0002 0.0050 ± 0.0005
ZrSiPr 2 h < LOD 0.013 0.14 ± 0.11 0.017 0.33 ± 0.12
Pr release 24 h < LOD < LOD 0.012 ± 0.0078 0.0046 ± 0.0056 0.046 ± 0.0017

Released/dissolved amount of elements per total amount of loaded material [μg/μg].

(test item) Exposure GMB PBS ASW ALF GST
Material period pH 7.4 pH 7.2 pH 6.5 pH 4.5 pH 1.7
ZrSiPr 2 h < LOD < LOD 0.00002 0.000009 < LOD
Zr release 24 h < LOD < LOD < LOD 0.00003 ± 0.000008 0.00002
ZrSiPr 2 h < blank 0.0003 ± 0.00008 < blank 0.0004 ± 0.00004 0.0003 ± 0.0001 
Si release 24 h < blank 0.00007 ± 0.00031  0.00009 ± 0.00003 0.00004 ± 0.00002 0.0006 ± 0.00006
ZrSiPr 2 h < LOD 0.0001 0.0014 ± 0.0012  0.0002 0.0035 ± 0.0012
Pr release 24 h < LOD < LOD 0.0015 ± 0.001 0.0006 ± 0.0007 0.0057 ± 0.0002

Elements transformed [mass %], equivalent to their percentage of the elemental content of the total amount of particles loaded; shown as average of triplicate samples in the different media. Blank values for each individual media and exposure period have been subtracted.

(test item) Exposure GMB PBS ASW ALF GST
Material period pH 7.4 pH 7.2 pH 6.5 pH 4.5 pH 1.7
ZrSiPr 2 h < LOD < LOD 0.0015 0.0009 < LOD
Zr release 24 h < LOD < LOD < LOD 0.0025 ± 0.0008 0.002
ZrSiPr 2 h < blank 0.027 ± 0.0079 < blank 0.036 ± 0.0038 0.027 ± 0.011
Si release 24 h < blank 0.0068 ± 0.031 0.0094 ± 0.0031 0.0039 ± 0.0019 0.063 ± 0.014
ZrSiPr 2 h < LOD 0.013 0.14 ± 0.12 0.018 0.35 ± 0.12
Pr release 24 h < LOD < LOD 0.15 ± 0.098 0.058 ± 0.070 0.57 ± 0.02

Total released/dissolved amount of elements per total amount of loaded material [μg/μg] in %.

 Exposure GMB PBS ASW ALF GST
Test item  time pH 7.4 pH 7.2 pH 6.5 pH 4.5 pH 1.7
ZrSiPr 24 h < LOD 0.007 ± 0.031 0.16 ± 0.10 0.065 ± 0.073 0.64 ± 0.03

Elements transformed [mass %], equivalent to the percentage of the released element compared to its amount within the amount of particles loaded.

(test item) Exposure GMB PBS ASW ALF GST
Material period pH 7.4 pH 7.2 pH 6.5 pH 4.5 pH 1.7
ZrSiPr 2 h < LOD < LOD 0.0035 ± 0.0004 0.0020 ± 0.004 < LOD 
Zr release 24 h < LOD < LOD < LOD 0.0058 ± 0.0018 0.0046 ± 0.0011
ZrSiPr 2 h < blank 0.17 ± 0.049 < blank 0.23 ± 0.024 0.17 ± 0.067
Si release 24 h < blank 0.042 ± 0.19 0.058 ± 0.019 0.024 ± 0.012 0.39 ± 0.039
ZrSiPr 2 h < LOD 0.34 3.6 ± 3.0 0.44 8.7 ± 3.1
Pr  release 24 h < LOD < LOD 3.7 ± 2.5 1.5 ± 1.8 14.5 ± 0.53

Applicant's summary and conclusion

Conclusions:
As highest dissolved Zr, Si and Pr concentrations were below 2.4 µg/L (ALF, 24h), 62.7 µg/L (GST, 24h) and 574 µg/L (GST, 24h), respectively, even at the highest loading of 0.1 g/L, referring to a solubility of 0.0024 %, 0.063 % and 0.574 %, respectively, the pigment is considered biologically inert.
Executive summary:

The bioaccessibility of zirconium praseodumium yellow zircon has been investigated experimentally in vitro by simulating dissolution under physiological conditions considered to mimic the most relevant exposure routes (oral, dermal and inhalation), as follows:


 


- Gamble’s solution (GMB, pH 7.4) which mimics the interstitial fluid within the deep lung under normal health conditions,


- Phosphate-buffered saline (PBS, pH 7.2), which is a standard physiological solution that mimics the ionic strength of human blood serum,


- Artificial sweat (ASW, pH 6.5) which simulates the hypoosmolar fluid, linked to hyponatraemia (loss of Na+ from blood), which is excreted from the body upon sweating,


- Artificial lysosomal fluid (ALF, pH 4.5), which simulates intracellular conditions in lung cells occurring in conjunction with phagocytosis and represents relatively harsh conditions and


- Artificial gastric fluid (GST, pH 1.5), which mimics the very harsh digestion milieu of high acidity in the stomach.


 


The dissolution of zirconium of the test item zirconium praseodumium yellow zircon is at a loading of 0.1 g/L in a range of <LOD (GMB, PBS, GST) and 1.5 µg/L (ASW) after 2 hours and below 2.4 µg/L (ALF) after 24 hours.


The dissolution of silicium of the test item zirconium praseodumium yellow zircon is at a loading of 0.1 g/L in a range of <blank (GMB, ASW) and 36.7 µg/L (ALF) after 2 hours and below 62.7 µg/L after 24 hours.


The dissolution of praesodymium of the test item zirconium praseodumium yellow zircon is at a loading of 0.1 g/L in a range of <LOD (GMB) and 348 µg/L (GST) after 2 hours and below 574 µg/L after 24 hours.


 


Therefore, zirconium praseodumium yellow zircon may reasonably be considered not bioaccessible.