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Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Objective of study:
other: Bioaccessibility
Qualifier:
no guideline followed
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
Species:
other: in vitro (simulated human body fluids)
Details on test animals and environmental conditions:
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).
Route of administration:
other: in vitro (simulated human body fluids)
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
Toxicokinetic parameters:
other: bioaccessibility
Remarks:
Dissolution of Zr was below 2.4 µg/L after 24 hours and Si was below 62.7 µg/L after 24 hours at pH 4.5. Dissolution of Pr from the test item was determined to be 348 µg/L (pH 1.7) after 2 hours and below 574 µg/L after 24 hours.

Zirconium praseodumium yellow zircon - ZrSiPr

BET-analysis:

The specific surface area, measured by BET-analysis is 0.52m²/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±.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
Conclusions:
Interpretation of results (migrated information): other: biologically inert
The dissolution of zirconium of the test item zirconium praseodumium yellow zircon is at a loading of 0.1g/L in a range of The dissolution of silicium of the test item zirconium praseodumium yellow zircon is at a loading of 0.1g/L in a range of The dissolution of praesodymium of the test item zirconium praseodumium yellow zircon is at a loading of 0.1g/L in a range of
Endpoint:
basic toxicokinetics, other
Remarks:
mass balance
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-03-01 to 2015-03-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Objective of study:
other: mass balance
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 5 male and 5 female Crl:CD(SD) rats were dosed orally with a single administration of zirconium praseodymium yellow zircon, vehicle (0.8 % aqueous hydroxyl propyl methylcellulose gel), or reference item (praseodymium chloride) via gavage. Clinical signs, mortality and body weight were recorded. Furthermore, urine and faeces of all animals were collected in metabolic cages after administration (sampling period: 0 - 24 hours, 24 - 48 hours, and 48 - 72 hours). Lastly, gross pathology was conducted and organ weights were determined.
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2014-05-14
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, keep dry in a tightly closed container
Radiolabelling:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The rat is a commonly used rodent species for toxicity studies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at dosing: males: 48 days; females: 53 days
- Weight at dosing: males: 234.7 - 263.7 g; females: 183.4 - 221.3 g
- Housing (exception: sampling period): kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm × 23 cm and a height of approx. 18 cm; bedding material: granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany)
- Diet (ad libitum): commercial ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): drinking water
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C ± 3 °C (maximum range)
- Relative humidity: 55% ± 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: zirconium praseodymium yellow zircon: 0.8% aqueous hydroxyl propyl methylcellulose gel; reference item (praseodymium chloride): water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
1) Zirconium praseodymium yellow zircon
The test item formulations were freshly prepared on the administration day by dissolving the test item in the vehicle to the appropriate concentrations. During and after preparation the formulation was stirred and the homogeneity of the suspension was checked by visual appraisal.
Administration volume: 10 mL/kg bw
The administration formulations were continuously agitated by stirring throughout the entire administration procedure.
The amount of the test item was adjusted to the animal's current body weight on the administration day.

2) Reference item (Praseodymium chloride (purity: 99.99 %)
The following dose was administered to the rats via gavage:
70 mg praseodymium chloride/kg bw
The test item formulations were freshly prepared on the administration day by dissolving the reference items in the vehicle to the appropriate concentrations. During and after preparation the formulation was stirred and the homogeneity of the suspension was checked by visual appraisal.
Administration volume: 10 mL/kg bw
The administration formulations were continuously agitated by stirring throughout the entire administration procedure.
The amount of the reference item was adjusted to the animal's current body weight on the administration day.


Duration and frequency of treatment / exposure:
single administration
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 males / 5 females
Control animals:
yes, concurrent vehicle
Positive control:
none
Details on study design:
- Dose selection rationale: the dose levels for this study have been selected after consultation with the Sponsor based on available toxicity data:
The oral LD50 value for the reference item was as follows:
The oral LD50 value for praseodymium chloride is not available; oral bioavailability of soluble Pr substance is given in the public domain with <1%.

The test item oral dose of 1000 mg/kg bw corresponds to the limit dose used in a separate 28-day oral toxicity study and is considered to be the maximum feasible dose. Based on the chemical composition of the test item, a dose of 1000 mg/kg bw of zirconium praseodymium yellow zircon equates to a dose of 40 mg Pr/kg bw, corresponding to a dose of 70 mg PrCl3/kg bw (anhydrous compounds).

The dose level for the reference item praseodymium chloride has been confirmed in a preliminary experiment (non-GLP) employing two animals per group (please also refere to IUCLID Section 7.1.1: k_Leuschner_2017_ in vivo). No toxicity was observed.
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine and faeces
- Time and frequency of sampling: all animals of the test item, vehicle and reference item groups were scheduled for urine and faeces sampling. After the single administration, the animals were kept in metabolism cages. Urine and faeces were collected in 3 fractions/animal (sampling periods: 0 - 24 hours, 24 - 48 hours, and 48 - 72 hours).
The urine and faeces weight per collection fraction and animal were determined upon removal of the sample fraction.
All samples were frozen at -20°C or colder and stored at this temperature until analysis.

OBSERVATIONS
- clinical signs: before and after dosing as well as regularly throughout the working day (7.30 a.m. to 4.30 p.m.) and on Saturdays and Sundays (8.00 a.m. to 12.00 noon; final check at approx. 4.00 p.m).
- mortality: early in the morning and again in the afternoon of each working day as well as on Saturdays and Sundays (final check at approx. 4.00 p.m).
- body weight: at the time of group allocation and on the day of administration

GROSS PATHLOLOHY / HISTOPATHOLOGY
- Necrospy and macroscopic inspection: on test day 4 (approx. 72 hours after the administration) the animals were dissected.
The animals were sacrificed, weighed, dissected, and inspected macroscopically.
All superficial tissues were examined visually and by palpation and the cranial roof was removed to allow observation of the brain, pituitary gland, and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole. The stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intra-abdominal lymph nodes, and accessory reproductive organs were recorded.

The weight of the following organs of all animals was determined: adrenal gland (2), brain, heart, kidney (2), liver, lungs, lymph nodes (cervical (1), mesenteric (1)), ovary (2), pituitary, prostate, spleen, testicle (2), thymus, and thyroid (1) (including parathyroids).
Paired organs were weighed individually and identified as left or right.

TEST ITEM FORMULATION ANALYSIS
Remaining administration formulations (approx. 5 mL) of each test and reference item that were mixed with a vehicle were stored at ≤-20°C until analysis (number of samples: 2).
Statistics:
The test item-treated and reference item-treated groups were compared statistically to the vehicle control group.
The following statistical method was used:
Multiple t-test based on DUNNETT, C. W. New tables for multiple comparisons with a control. Biometrics, 482-491 (Sept 1964): body weight / relative and absolute organ weights (p ≤ 0.01 and p ≤ 0.05)
Preliminary studies:
Please refer to the field "Details on study design" above.
Details on absorption:
Urinary excretion for Pr and Zr was negligible and below 0.00001%.
Details on excretion:
Animals that received 1000 mg pigment /kg bw excreted 102% Pr and 74.3% Zr of the administered dose via urine and faeces during the first three days after exposure (mean for 10 animals). Within the first 24 hours approximately 100% of Pr and 74.3% Zr were excreted via faeces as largest fraction. Further 1.75% and 0.14% (Pr) were excreted via faeces on the second and third day.
Urinary excretion for Pr was negligible and below 0.00001%.

CLINICAL SIGNS, MORTALITY, BODY WEIGHT, AND GROSS PATHOLOGY

- none of the rats treated once orally with 1000 mg Zirconium praseodymium yellow zircon/kg bw or 70 mg Praseodymium chloride/kg bw showed any changes in behaviour or external appearance.

- faeces of control and test and reference item-treated animals were normally formed.

- none of the rats died prematurely.

- individual body weights ranged from 234.7 to 263.7 g for the male animals and from 183.4 to 221.3 g for the female animals on test day 1 and were within the expected range.

- no changes were noted for the animals treated once orally with 1000 mg Zirconium praseodymium yellow zircon/kg bw or 70 mg Praseodymium chloride/kg bw at macroscopic inspection at necropsy.

- no test or reference item-related changes in relative and absolute organ weights were noted for the male and female rats treated with 1000 mg Zircon praseodymium yellow zircon/kg bw or 70 mg Praseodymium chloride/kg bw.

Statistically significant differences (p ≤ 0.05) in relative and absolute organ weights compared to the control which are not considered to be test item-related are given below:

females (reference item; test day 4): increased relative left kidney weight

males (test item; test day 4): increased absolute left testis weight

Conclusions:
Overall the mass balances for Pr and Zr is essentially complete and indicates that the element contained in the pigment "Zirconium praseodymium yellow zircon", present as Pr3+ and Zr4+ are not absorbed in the gastrointestinal tracts to any significant extent, but pass the animal effectively unchanged. 
Executive summary:

Animals that received 1000 mg pigment /kg bw excreted 102% Pr and 74.3% Zr of the administered dose via urine and faeces during the first three days after exposure (mean for 10 animals). Within the first 24 hours approximately 100% of Pr and 74.3% Zr were excreted via faeces as largest fraction. Further 1.75% and 0.14% (Pr) were excreted via faeces on the second and third day.

Urinary excretion for Pr was negligible and below 0.00001%.

Approximately 67.5 % of the administered dose for Pr and 72.8% for Zr were determined indirectly by weighing the undigested pigment which could visually be observed and isolated in/from the faeces residues of the 0-24 h samples after the digestion procedure in highly acidic media in an ultrasonic bath. Analytical verification of the elements Pr and Zr in the isolated pigment are currently under finalisation and will be available soon.

Samples for the 28-48 and 48-72 hour intervals did not show any obvious pigment residues (see pictures attached to this robust study summary “K_Leuschner_2017_mass balance”) which is in line with the observations received from other pigment studies that the majority of the pigment doses are excreted within the first 24 hours.

Since Zr4+has a very low absorbance ability within the g.i. tract (<1%) a hypothesis that this fraction was contained in the exsanguinated blood of the animals or has distributed to the organs seems not appropriate.

Overall the mass balances for Pr and Zr is essentially complete and indicates that the element contained in the pigment "Zirconium praseodymium yellow zircon", present as Pr3+ and Zr4+ are not absorbed in the gastrointestinal tracts to any significant extent, but pass the animal effectively unchanged. 

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-03-09 to 2015-03-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Objective of study:
toxicokinetics
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Version / remarks:
2010-07-22
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2014-05-14
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, kept dry and stored in a tightly closed container.
Radiolabelling:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The rat is a commonly used rodent species for toxicity studies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at dosing: males: 45 days; females: 52 days
- Weight at dosing: males: 206 - 233 g; females: 192 - 214 g
- Housing: kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 x 23 cm and a height of approx. 18 cm; bedding material: granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany)
- Diet (ad libitum): commercial ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): drinking water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- Relative humidity: 55% ± 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
other: zirconium praseodymium yellow zircon: oral (gavage); reference item (praseodymium chloride): oral (gavage) and intravenously injected
Vehicle:
other: zirconium praseodymium yellow zircon: 0.8% aqueous hydroxyl propyl methylcellulose gel; reference item (praseodymium chloride): water (gavage) or water for injection (intravenously injected)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
1) Zirconium praseodymium yellow zircon:
The test item formulation was freshly prepared on the administration day by dissolving the test item in the vehicle to the appropriate concentrations.

2) Reference item (Praseodymium chloride (purity: 99.99 %))
The reference item formulation was freshly prepared on the administration day by dissolving the reference item in the vehicle to the appropriate concentrations.
The following dosages of the reference item were administered:
a) oral administration: 70 mg praseodymium chloride/kg bw
b) intravenous administration: 0.7 mg praseodymium chloride/kg bw

The administration formulations were continuously agitated by stirring throughout the entire administration procedure.
Administration volume (oral administration / intravenous administration): 10 mL/kg bw

Injection speed (intravenous adminsitration): dose per approx. 15 seconds

The amount of test item and reference item was adjusted to each animal's current body weight on the administration day.
Duration and frequency of treatment / exposure:
single administration
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 males / 5 females
Control animals:
no
Positive control:
none
Details on study design:
- Dose selection rationale: the dose levels for this study had been selected after consultation with the Sponsor based on available toxicity data:
The oral LD50 value for the reference item was as follows:
The oral LD50 value for praseodymium chloride is not available in the public domain; oral bioavailability of soluble Pr substance is given in the public domain with <1%.

The test item oral dose of 1000 mg/kg bw corresponds to the limit dose used in a separate 28-day oral toxicity study, which is considered the maximum feasible dose. Based on the chemical composition of the test item, a dose of 1000 mg/kg b.w. of zirconium praseodymium yellow zircon equates to a dose of 40 mg Pr/kg, corresponding to a dose of 70 mg PrCl3/kg bw. (anhydrous compounds).

The dosage for the reference item administered by intravenous injection was set 1% of the dose of the test item on a stoichiometric basis for each metal, thereby lowering the dose for reasons of tolerability of the test animals. This equates to a dose of 0.4 mg Pr/kg bw, corresponding to a dose of 0.7 mg PrCl3/kg bw (anhydrous compounds).

The dose levels for the reference item praseodymium chloride (oral (gavage) and intravenously injected) have been confirmed in a preliminary experiment (non-GLP) employing two animals per group. No toxicity was observed.
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: plasma
- Time and frequency of sampling: blood was collected 0 (predose), 1, 2, 4, 8, 12, 24, 48, and 72 hours after administration. The whole blood samples were cooled using an IsoTherm-Rack system until centrifugation. Immediately after centrifugation, the plasma was frozen at ≤-20°C, and stored at this temperature until analysis.
In addition, 4 mL pooled blank plasma (approx. 2 mL per sex) were obtained from spare animals.

A toxicokinetic evaluation of the data on zirconium praseodymium yellow zircon plasma levels will be performed. A non-compartment model will be employed. The following parameters will be determined, if possible:
AUC0-inf = extrapolated area from zero to infinity
AUC0-t last = extrapolated area from time zero to the last quantifiable plasma concentration >LOQ
Kel = elimination rate constant
t½ = elimination half-life

Cmax values will be the highest measured plasma concentrations, and tmax values will be the time points of highest plasma concentrations.

Elimination rate constants (Kel) and plasma elimination half-lives (t½) will be calculated by linear regression analysis of the log/linear portion of the individual plasma concentration-time curves (c = concentration, t = time).

Area under the curve (AUC) values will be calculated using the linear trapezoidal method and extrapolated to infinite time by dividing the last measurable plasma concentration by the elimination rate constant. Plasma concentrations at time zero will be taken to be those at the first blood sampling time.

Furthermore, AUC0-t last-value will be calculated according to the linear trapezoidal rule. Values below or at the limit of quantification (LOQ) will be excluded from the calculation.

In addition, the bioavailability will be calculated for the mixture.

OBSERVATIONS
- clinical signs: before and after dosing as well as regularly throughout the working day (7.30 a.m. to 4.30 p.m.) and on Saturdays and Sundays (8.00 a.m. to 12.00 noon; final check at approx. 4.00 p.m).
- mortality: early in the morning and again in the afternoon of each working day as well as on Saturdays and Sundays (final check at approx. 4.00 p.m).
- body weight: at the time of group allocation and on the administration day.

TEST ITEM FORMULATION ANALYSIS
The remaining administration formulations of each test and reference item that was mixed with a vehicle were stored at ≤- 20°C (approximately 5 mL per formulation, in total 3 samples).
Statistics:
The reference item-treated group (oral administration) was compared to the test item treated group.
The following statistical method was used:
STUDENT's t-test: body weight (p ≤ 0.01 and p ≤ 0.05)
The following limits were used:
p = 0.05 / 0.01 about t = 2.3060 / 3.3554
(for 8 degrees of freedom)
Preliminary studies:
Please refer to the field "Details on study design" above.
Toxicokinetic parameters:
other: bioavailability
Remarks:
The relative bioavailability of orally administered pigment was determined in relation to a soluble Pr3+compound (PrCl3) injected i.v... Result: relative bioavailability 0.000011% (Pr).
Bioaccessibility testing results:
For Pr an absolute bioavailability of 0.000055%% (mean m/f) was calculated from soluble PrCl3 following oral administration compared to intravenous administration, and a relative bioavailability of approximately 0.000011% (mean m/f) for Pr present in the pigment.

LOCAL TOLERANCE (REFERENCE ITEM; INTRAVENOUS ADMINISTRATION)

No signs of local intolerance reactions were noted at the injection sites of the rats treated once intravenously with 0.7 mg praseodymium chloride/kg bw.

CLINICAL SIGNS, MORTALITY, AND BODY WEIGHT

Zirconium praseodymium yellow zircon:

None of the rats treated once orally with 1000 mg Zircon praseodymium yellow zircon/kg bw showed any changes in behaviour or external appearance.

The faeces of the animals were normally formed.

None of the rats died prematurely.

Body weight of all animals was in the expected range on test day 1.

Reference item (oral administration):

None of the rats treated once orally with 70 mg Praseodymium chloride/kg bw showed any changes in behaviour or external appearance.

The faeces of the animals were normally formed.

None of the rats died prematurely.

Body weight of all animals was in the expected range on test day 1.

Reference item (intravenous administration):

None of the rats treated once intravenously with 0.7 mg Praseodymium chloride/kg bw showed any changes in behaviour or external appearance.

The faeces of the animals were normally formed.

None of the rats died prematurely.

Body weight of all animals was in the expected range on test day 1.

Conclusions:
In a relative bioavailability study, the relative bioavailability of orally administered pigment was calculated relative (0.000011% (Pr)) in relation to a soluble Pr3+ compound (PrCl3) injected i.v..
Executive summary:

In conclusion, the oral relative bioavailability of the pigment "Zirconium praseodymium yellow zircon" can be assumed to be negligible. The absolute bioavailability of orally administered soluble Pr3+compound (PrCl3) in relation to soluble Pr3+compound (PrCl3), injected i.v. was calculated to be 0.000055% (Pr). A relative bioavailability of orally administered pigment was calculated as 0.000011% (Pr).

Description of key information

The in-vitro and in-vivo experiments described above are in very good agreement with regards to the negligible level of bioavailability of the elements Zr and Pr contained in the pigment.

(1)   In in-vitro dissolution experiments in five different artificial physiological media, dissolved Zr and Pr concentrations from this pigment were below 2.4 μg/L, 350 μg/L even at the highest loading of 0.1g/L, corresponding to a solubility of less than 0.4%,

(2)   In a 28-day oral toxicity study with 1,000 mg/kg pigment no increase in Zr and Pr plasma and urine concentrations were observed when sampled at the end of the 28-day exposure period. From a final dose of 1,000 mg/kg of the pigment that the animals received on the last day of the study, only cumulated relative amounts of << 0.001 % (m/f) were found in the terminal 24-h urine collection period.

(3)   In a mass balance study with a single oral dose of 1,000 mg/kg of the pigment, 102% Pr of the dose and 74.3% Zr were excreted via faeces within 3 days, with only <0.00001% Pr and < 0.002% Zr of the dose being excreted via urine at the same time.

(4)   In a bioavailability study, the absolute (0.000055% (Pr)) and relative (0.000011% (Pr)) bioavailability of orally administered pigment was calculated in relation to a soluble Pr3+compound (PrCl3), injected i.v..

Comparing the findings ofin-vitrodissolution testing (1) within-vivoresults (2-4), thein-vivodata consistently demonstrates slightly lower bioavailability. This is in agreement with the general understanding thatin-vitroexperiments in simulated gastric juice provide a conservative estimate of actual (in-vivo) bioavailability.

In conclusion, the oral relative bioavailability of the pigment "Zirconium praseodymium yellow zircon" can be assumed to be negligible, as demonstrated in three independent in-vivo studies in rats yielding very comparably results supported by anin-vitrodissolution experiment in five different artificial physiological media.

A rounded value of <<0.01% for oral absorption can be taken forward from (i) terminal urine/plasma sampling in a study involving 28 repeated oral doses of 1,000 mg pigment/kg bw/d (<<0.001% for Pr) and (ii) a mass balance study involving a single dose of 1,000 mg pigment/kg bw (0.00003% for Pr and <<0.00001% for Zr).

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

The toxicity data in this registration dossier refer explicitly to the pigment Zirconium praseodymium yellow zircon and document its negligible bioavailability and the complete lack of any human health hazard. Experiments on the bioavailability of the pigment are summarised and discussed in this section.

Summary ofin-vitrobioaccessibility experiments (Herting, Wallinder, 2010)

The chemical and physiological properties of the pigment Zirconium praseodymium yellow zircon are characterised by inertness because of the specific synthetic process (calcination at high temperatures, approximately 1000°C), rendering the substance to be of a unique, stable crystalline structure in which all atoms are tightly bound and not prone to dissolution in environmental and physiological media. This manufacturing process leads to a very low bioaccessibility of the elements contained in the pigment. This 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:

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

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

3.) 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,

4.) 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

5.) artificial gastric fluid (GST, pH 1.7), which mimics the very harsh digestion milieu of high acidity in the stomach.

The dissolution of zirconium from the test item Zirconium praseodymium yellow zircon was in a range of < LoD and 1.5 μg/L (pH 6.5) after 2 hours and in a range of < LoD and 2.4 μg/L (pH 4.5) at a loading of 0.1g/L after 24 hours. Further, the dissolution of praseodymium from the test item Zirconium praseodymium yellow zircon was in a range of < LoD (pH 7.4) and 348 μg/L (pH 1.7) after 2 hours and in a range of < LoD (pH 7.2 and 7.4) and 574 μg/L (pH 1.7) at a loading of 0.1g/L after 24 hours. Dissolution of Si was determined to be < blank (pH 6.5 and 7.4) and 36.7 μg/L (pH 4.5) after 2 hours and in a range of < blank (pH 7.4) and 62.7 μg/L (pH 1.7) at a loading of 0.1g/L after 24 hours.

In conclusion, since the dissolved Zr, Pr and Si concentrations from this pigment were below 2.4 μg/L, 350 μg/L (2-hour value most relevant due to gastric passage duration of about 2-3 ours) and 26.8 μg/L (2-hour gastric passage) even at the highest loading of 0.1g/L, corresponding to a solubility of less than 0.4%, this pigment may reasonably be considered biologically inert.

 

Toxicokinetic screening data from a 28 day repeated dose oral toxicity study (Leuschner, 2017)

In a 28 day repeated dose toxicity study, male and female rats were given a daily dose of the pigment "Zirconium praseodymium yellow zircon" of 1,000 mg/kg bw/day via gavage. Individual urine samples were collected from all animals prior to sacrifice in one cumulated 24-h fraction/animal after the last oral application, and blood samples were collected from each animal upon sacrifice. The plasma and urine samples were analysed for total zirconium and praseodymium content.

The uptake of zirconium and praseodymium during a 24 hour urine and plasma sampling period was demonstrated to be negligible considering that <<0.001% of the dose was excreted via urine for both metals, mirrored by either minimal or no increases in blood plasma concentrations.

The zirconium and praseodymium concentrations of the 24h-urine samples, collected during the day before final sacrifice, ranged from: 0.005 – 0.015 μg/L urine (mean: 0.009 ± 0.004) and 0.001 – 0.019 μg/L urine (mean: 0.011 ± 0.015) for Pr and from 0.261 – 0.648 μg/L urine (mean: 0.376 ± 0.155) and 0.118 – 0.434 μg/L urine (mean: 0.267 ± 0.14) for Zrforthe male and female animals of the control group, respectively.

For the dosed group, the concentrations were0.012 – 0.03 μg/L urine (mean: 0.023 ± 0.007) and 0.015 – 0.102 μg/L urine (mean: 0.048 ± 0.036)for Pr and 0.088 – 0.287 μg/L urine (mean: 0.22 ± 0.08) and 0.184 – 0.61 μg/L urine (mean: 0.396 ± 0.159) for Zrfor the male and female animals, respectively.

Following a subtraction of the background urinary element excretion (control group), and considering the excreted urine volume (mean 21.4 mL (m) and 14.6 mL (f)) and the body weight of the animals at the end of the study (mean 369.2 g (m) and 223.92 g (f)), the following conclusion can be made:

From a final dose of 1,000 mg/kg of the pigment that the animals received on the last day of the study, only cumulated relative amounts of 0.000015 % (m) or 0.0000037% (f) were found in the terminal 24-h urine collection period.

Summary of comparative Mass-Balance Study (Leuschner 2017a):

In a comparative mass balance study involving oral dosing of (i) the inorganic pigment "Zirconium praseodymium yellow zircon" and (ii) a soluble salt of the element contained therein (Pr3+), the gastrointestinal absorption as well as urinary and faecal excretion were compared, plus consideration of dietary „background“ intake/excretion via a vehicle-dosed control. For details, please refer to the corresponding robust study summary.

In brief, 10 (5m/5f) animals per group received a single oral dose of 1000 mg/kg of the pigment or70 mg PrCl3,(corresponding to 40 mg/kg Pr). A third group served as vehicle treated control. Animals were individually housed in metabolic cages and daily samples of urine and faeces were collected for three days. All samples were analysed for praseodymium. The averaged “background” excretion via urine and faeces of the control animals was subtracted from the amounts excreted by the dosed animals, and a mass balance was calculated.

Animals that received 40 mg Pr/Kg bw (administered as PrCl3) excreted 91.5 % (Pr) of the administered dose (as mean, male and female animals) via urine (0.00015%) and faeces (91.5%) during the first three days after exposure.The largest fraction (85.49 % Pr) was excreted via faeces and urine already within the first 24h.

Animals that received 1000 mg pigment /kg bw excreted 102% Pr and 74.3% Zr of the administered dose via urine and faeces during the first three days after exposure (mean for 10 animals). Within the first 24 hours approximately 100% of Pr and 74.3% Zr were excreted via faeces as largest fraction. Further 1.75% and 0.14% (Pr) were excreted via faeces on the second and third day.

Urinary excretion for Pr was negligible and below 0.00001%.

Approximately 67.5 % of the administered dose for Pr and 72.8% for Zr were determined indirectly by weighing the undigested pigment which could visually be observed and isolated in the faeces residues of the 0-24 h samples after the digestion procedure in highly acidic media in an ultrasonic bath. Samples for the 28-48 and 48-72 hour intervals did not show any obvious pigment residues (see pictures attached to the robust study summary “K_Leuschner_2017_mass balance”) which is in line with the observations received from other pigment studies that the majority of the pigment doses are excreted within the first 24 hours.

Since Zr4+has a very low absorbance ability within the g.i. tract (<1%) a hypothesis that this fraction was contained in the exsanguinated blood of the animals or has distributed to the organs seems not appropriate.

Overall the mass balances for Pr is essentially complete and indicates that the element contained in the pigment "Zirconium praseodymium yellow zircon", present as Pr3+is not absorbed in the gastrointestinal tracts to any significant extent, but pass the animal effectively unchanged. 

Summary of relative bioavailability study (Leuschner 2017b):

A relative bioavailability study involving serum kinetics over a period of 72 hours p. a. involving an i. v. dosing of a soluble Pr reference substance (praseodymium chloride) compared to single oral doses of the same substances and the pigment was performed. For details, please refer to the corresponding robust study summary. In brief, 10 animals (5m/5f) per group received single doses of (1) 0.7 mg/kg PrCl3intravenously, (2) 70 mg/kg PrCl3, via oral gavage, and (3) 1000 mg/kg of the pigment via oral gavage.

Blood samples were taken at 0, 1, 2, 4, 8, 12, 24, 48 and 72 hours post exposure and blood plasma samples were prepared and analysed for the element Pr.

Cmax-levels in plasma of 4.23 µg Pr/g and 4.40 µg Pr/g, were noted 1 hour (tmaxas mean m/f) after intravenous administration of a mixture of 0.7 mg/kg PrCl3for the male and female rats on test day 1, respectively.

Furthermore, Cmax-levels of 0.0002 µg Pr/g and 0.0001 µg Pr/g were noted ~3.2, or 1.2 hours (tmaxas mean m/f) after oral administration of 70 mg/kg PrCl3for the male and female rats on test day 1, respectively.

Lastly, Cmax-levels of 0.0002 µg Pr/g and 0.0001 µg Pr/g, were noted 0 - 1 hour (tmaxas mean m/f) after oral administration of 1000 mg pigment/kg for the male and female rats on test day 1, respectively. For comparison, the average (n=30) concentration of praseodymium in plasma taken before exposure at t=0 h was 6.8*10-6µg Pr/g.

The plasma concentrations declined post dosing with an elimination half-life ranging from 2 to 25.5 hours for Pr.

For Pr an absolute bioavailability of 0.000048/0.000062% (m/f) calculated from soluble PrCl3following oral administration compared to intravenous administration, and a relative bioavailability of approximately 0.000007/0.000015% (m/f) for Pr present in the pigment could be calculated.

In sum, experimentally determined plasma kinetic values for Pr are very consistent and the very low relative bioavailabilities of Pr from the pigment demonstrates that the pigment can be considered inert without any systemic hazard potential for human health.

Overall conclusion:

The in-vitro and in-vivo experiments described above are in very good agreement with regards to the negligible level of bioavailability of the elements Zr and Pr contained in the pigment.

(1)   In in-vitro dissolution experiments in five different artificial physiological media, dissolved Zr and Pr concentrations from this pigment were below 2.4 μg/L, 350 μg/L even at the highest loading of 0.1g/L, corresponding to a solubility of less than 0.4%,

(2)   In a 28-day oral toxicity study with 1,000 mg/kg pigment no increase in Zr and Pr plasma and urine concentrations were observed when sampled at the end of the 28-day exposure period. From a final dose of 1,000 mg/kg of the pigment that the animals received on the last day of the study, only cumulated relative amounts of << 0.001 % (m/f) were found in the terminal 24-h urine collection period.

(3)   In a mass balance study with a single oral dose of 1,000 mg/kg of the pigment, 32.5% Pr of the dose and 1.5% Zr were excreted via faeces within 3 days, with only <0.00001% Pr and0% Zr of the dose being excreted via urine at the same time.

(4)   In a bioavailability study, the absolute (0.000055% (Pr)) and relative (0.000011% (Pr)) bioavailability of orally administered pigment was calculated in relation to a soluble Pr3+compound (PrCl3), injected i.v..

Comparing the findings ofin-vitrodissolution testing (1) within-vivoresults (2-4), thein-vivodata consistently demonstrates slightly lower bioavailability. This is in agreement with the general understanding thatin-vitroexperiments in simulated gastric juice provide a conservative estimate of actual (in-vivo) bioavailability.

In conclusion, the oral relative bioavailability of the pigment "Zirconium praseodymium yellow zircon" can be assumed to be negligible, as demonstrated in three independent in-vivo studies in rats yielding very comparably results supported by anin-vitrodissolution experiment in five different artificial physiological media.

A rounded value of <<0.01% for oral absorption can be taken forward from (i) terminal urine/plasma sampling in a study involving 28 repeated oral doses of 1,000 mg pigment/kg bw/d (<<0.001% for Pr) and (ii) a mass balance study involving a single dose of 1,000 mg pigment/kg bw (0.00003% for Pr and <<0.00001% for Zr).

 

Absorption rate - oral: << 0.01%