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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 or test system 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
Type:
other: Bioaccessibility
Results:
Dissolution of Si from the test item:

Cristobalite - SiFe

BET-analysis:

The specific surface area, measured by BET-analysis is 4.21 m²/g.It should be underlined that this specific surface area is measured by nitrogen absorption and includes also the surface of surface pores.

Metal analysis:

Particles of SiFe dissolved to a very low extent (Si+Fe) (GMB 0.43 %, PBS 0.43 %,ASW 0.0056 %, ALF 0.059 %, GST 0.076 %,after 24 hours of exposure). Si was predominantly released at neutral pH (GMB pH 7.4 and PBS pH 7.2) whereas Fe primarily was released at more acidic pH (ALF pH 4.5, GST pH 1.7).

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  
SiFe    2 h   96.6±30.9   34.9±16.1 4.1±5.0 < blank 6.6±10.0
Si release  24 h   406±35.9  413±34.7  < blank 25.8±19.5    12.1±1.7
SiFe  2 h   4.8±2.1 4.8±1.3 13.4±2.0 12.2±8.9 12.9±1.0
Fe release  24 h   11.2±0.9 6.7±1.4 5.3±1.7 31.4±2.5 62.6±4.7

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  
SiFe  2 h   0.012±0.0040 0.0043±0.0021 0.0005±0.0007  < blank 0.0008±0.0012
Si release    24 h   0.0041±0.0003 0.0042±0.0005 < blank 0.0003±0.0002 0.0001±0.00002
SiFe   2 h   0.0006±0.0003 0.0006±0.0002 0.0016±0.0002 0.0015±0.0011 0.0016±0.0001
Fe release    24 h   0.0001±0.00001 0.0001±0.00002 0.0001±0.00002 0.0003±0.00002 0.0006±0.0001

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  
 SiFe   2 h   0.001±0.0003 0.0004±0.0002 0.00004±0.00006   < blank 0.00007±0.00011
 Si release    24 h   0.0042±0.0003 0.0043±0.0005 < blank 0.0003±0.0002 0.0001±0.00002
 SiFe  2 h   0.00005±0.00002 0.00005±0.00001 0.0001±0.00002 0.0001±0.0001 0.0001±0.00001
 Fe release    24 h   0.0001±0.00001 0.00007±0.00002 0.00006±0.00002 0.0003±0.00002 0.0006±0.00006

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  
 SiFe   2 h   0.099±0.033 0.036±0.017 0.0044±0.0055 < blank 0.0069±0.011
 Si release    24 h   0.42±0.030 0.43±0.05 < blank 0.027±0.020   0.012±0.0022  
 SiFe   2 h   0.0050±0.0023 0.0049±0.0013 0.014±0.0016 0.013±0.0093 0.013±0.0010
Fe release    24 h   0.012±0.001 0.0069±0.0017 0.0056±0.0018 0.033±0.0020 0.064±0.0057

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  
 SiFe   24 h   0.43±0.03 0.43±0.05 0.0056±0.0018 0.059±0.022 0.076±0.008

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  
 SiFe   2 h   0.25±0.083 0.090±0.043 0.011±0.014 < blank 0.017±0.026
Si release    24 h   1.0±0.075 1.1±0.12 < blank 0.066±0.050 0.031±0.0055
 SiFe   2 h   0.053±0.024 0.052±0.014 0.14±0.017 0.13±0.098 0.14±0.0011
 Fe release    24 h   0.12±0.001 0.073±0.018 0.060±0.019 0.34±0.021 0.67±0.060
Conclusions:
The dissolution of silicium of the test item reaction mass of fumes, silica and diiron trioxide is at a loading of 0.1g/L in a range of The dissolution of iron of the test item reaction mass of fumes, silica and diiron trioxide is at a loading of 0.1g/L in a range of <4.8 (pH 7.4) and 13 µg/L (pH 6.5) after 2 hours and below 63 µg/L after 24 hours.
In conclusion, since the dissolved Si and Fe concentrations from this pigment were below 430 μg/L even at the highest loading of 0.1g/L, corresponding to a solubility of less than 0.45 %, this pigment may reasonably be considered biologically inert.
Endpoint:
basic toxicokinetics, other
Remarks:
mass balance
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-03-06 to 2015-03-09
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:
according to guideline
Guideline:
OECD Guideline 417 (Toxicokinetics)
Version / remarks:
2010-07-22
Deviations:
no
Principles of method if other than guideline:
Groups of 5 male and 5 female Crl:CD(SD) rats were dosed orally with a singel administration of reaction mass of fumes, silica and diiron trioxide, vehicle (0.8 % aqueous hydroxyl propyl methylcellulose gel), or reference item (iron (III) citrate monohydrate) 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. QA statement)
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 species was selected for this study design because the rat is a commonly used rodent species for toxicity studies.
Sex:
male/female
Details on test animals or test system 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: 230.1 - 258.1 g; females: 175.4 - 206.1 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: 9 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: Reaction mass of fumes, silica and diiron trioxide: 0.8 % aqueous hydroxyl propyl methylcellulose gel; reference item (iron (III) citrate monohydrate): tap water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Reaction mass of fumes, silica and diiron trioxide:
The test item was suspended in its vehicle to the appropriate concentration freshly on the day of administration.
The administration formulations were continuously agitated by stirring throughout the entire administration procedure.
Administration volume: 10 mL/kg bw
The amount of the test item was adjusted to the animal's current body weight on the administration day.

Reference item (iron (III) citrate monohydrate; assay: 18 % Fe):
One day before administration, the reference item and the appropriate vehicle were heated to 70°C and stirred at 50°C for approx. 3 hours until the reference item had completely dissolved. The administration formulation was stirred overnight (approx. 12 hours) at room temperature.
The administration formulations were continuously agitated by stirring throughout the entire administration procedure.
Administration volume: 10 mL/kg bw
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 / concentration:
5 males / 5 females
Control animals:
yes, concurrent vehicle
Positive control reference chemical:
none
Details on study design:
- Dose selection rationale: the dose levels for this preliminary 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:
iron citrate monohydrate: >2000 mg/kg bw
Furthermore, oral bioavailability of soluble Fe substances is given in the public domain with 1 to 26% (Fe).

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 reaction mass of fumes, silica and diiron trioxide/kg bw equates to doses of 94.4 mg Fe/kg bw (corresponding to 444 mg iron(III) citrate monohydrate/kg bw).

The dose level for the reference item has been confirmed in a preliminary experiment (non-GLP) employing two animals (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 and 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, intraabdominal lymph nodes, and accessory reproductive organs were recorded.

The weight of the following organs 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 was negligible and below 0.016% Fe.
Details on excretion:
Animals that received 1000 mg pigment /kg bw excreted 110.7% Fe 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 98.1% of Fe were excreted via faeces as largest fraction. Further 11.1% and 1.5% (Fe), were excreted via faeces on the second and third day. Urinary excretion was negligible and below 0.016% Fe.

CLINICAL SIGNS, MORTALITY, BODY WEIGHT, GROSS PATHOLOGY

Vehicle control group:

- no signs of systemic intolerance was observed

- faeces of control animals were normally formed.

- none of the rats died prematurely.

- individual body weights ranged from 233.1 to 248.1 g for the male animals and from 180.4 to 206.1 g for the female animals on test day 1 and were within the expected range.

- no pathological findings were recorded

 

Reaction mass of fumes, silica and diiron trioxide:

- none of the rats treated with the test item showed any changes in behaviour or external appearance.

- faeces of test item-treated animals were normally formed.

- none of the rats died prematurely.

- individual body weights ranged from 234.8 to 255.1 g for the male animals and from 178.7 to 201.5 g for the female animals on test day 1 and were within the expected range.

- no changes were noted for the animals treated with the test item at macroscopic inspection at necropsy.

- no test item-related changes in relative and absolute organ weights were noted for the male and female rats treated with the test item.

 

Reference item:

- none of the rats treated with the reference item showed any changes in behaviour or external appearance.

- faeces of reference item-treated animals were normally formed.

- none of the rats died prematurely.

- individual body weights ranged from 230.1 to 258.1 g for the male animals and from 175.4 to 200.1 g for the female animals on test day 1 and were within the expected range.

- no changes were noted for the animals treated with the reference item at macroscopic inspection at necropsy.

- no reference item-related changes in relative and absolute organ weights were noted for the male and female rats treated with the reference item.

Conclusions:
The mass balances for Fe is essentially complete and indicatess that the element Fe contained in the pigment "Reaction mass of fumes, silica and diiron trioxide", present as Fe3+, is not absorbed in the gastrointestinal tracts to any significant extent, but pass the animal effectively unchanged.

Iron is part of the diet that the animals received during the dosing and excretion period which may be considered with respect to the calculated mass balance of >100%. Assumed that the animals consumed approximately 10% of their individual bodyweight as diet, an additional "dosing" of Fe of about 3.86 mg/animal (average of 20 animals, male and female) seems reasonable which increases the total dose by 18.96%. The obtained results are considered to be realistic.

In total, the mass balances for iron is essentially complete and indicates that the element iron contained in the pigment " Reaction mass of Fumes, silica and diiron trioxide ", present as iron(III), is not absorbed in the gastrointestinal tracts to any significant extent, but pass the animal effectively unchanged. It is not absolutely clear whether the determination of ca. 0.016% Fe in urine during the first day of exposure is a result of Fe absorbed from the pigment and is presumably caused by bioavailable Fe from the diet.

Based on the fact that silica shows a very poor oral bioavailability in animals and humans (EFSA, 2018)* and due to the high not test item related background levels in organs, it was decided not to include a silica quantification in the mass balance study.

As detailed above, the pigment Reaction mass of Fumes, silica and diiron trioxide is almost exclusively excreted via faeces, leaving a minute amount of iron and silica becoming systemically available. In the mass-balance study summarised above, << 0.016% of the iron and silica was excreted via urine, i.e. was systemically available.

Executive summary:

Animals that received 1000 mg pigment /kg bw excreted 110.7% Fe 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 98.1% of Fe were excreted via faeces as largest fraction. Further 11.1% and 1.5% (Fe), were excreted via faeces on the second and third day.´

Urinary excretion was negligible and below 0.016% Fe.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-03-30 to 2015-04-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Objective of study:
toxicokinetics
Qualifier:
according to guideline
Guideline:
OECD Guideline 417 (Toxicokinetics)
Version / remarks:
2010-07-22
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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 species was selected for this study design because the rat is a commonly used rodent species for toxicity studies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at administration: males: 7 weeks; females: 6 or 8 weeks
- Weight at administration: males: 218 - 248 g; females: 163 - 205 g
- Housing: kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 × 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 or 6 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: Reaction mass of fumes, silica and diiron trioxide: oral (gavage); Reference item (iron (III) citrate monohydrate): oral (gavage) and intravenously injected
Vehicle:
other: Reaction mass of fumes, silica and diiron trioxide: 0.8 % aqueous hydroxyl propyl methylcellulose gel; reference item (iron (III) citrate monohydrate): water (oral administration) or 0.9 % NaCl solution (intravenous administration)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Reaction mass of fumes, silica and diiron trioxide:
The test item was suspended in its vehicle to the appropriate concentration freshly on the administration day.

Reference item (iron (III) citrate monohydrate; assay: 18 % Fe):
One day before administration, the reference item and the appropriate vehicle were heated to 70°C and stirred at 50°C for approx. 3 hours until the reference item had completely dissolved. The administration formulations were stirred overnight (approx. 12 hours) at room temperature.

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

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

The amount of test item and 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 / concentration:
5 males / 5 females
Control animals:
no
Positive control reference chemical:
none
Details on study design:
- Dose selection rationale: the dose levels for this preliminary 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:
iron citrate monohydrate: >2000 mg/kg bw
Furthermore, oral bioavailability of soluble Fe substances is given in the public domain with 1 to 26% (Fe).

The test item oral dose of 1000 mg/kg bw corresponds to the limit dose used in a separate 28d 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 bw of reaction mass of fumes, silica and diiron trioxide equates to a dose of 94.4 mg Fe/kg bw (corresponding to 444 mg iron(III) citrate monohydrate/kg bw).

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 doses of 0.94 mg Fe/kg bw (corresponding to 4.5 mg iron(III) citrate monohydrate/kg bw).

The dose levels for the reference item Iron (III) citrate monohydrate (oral and intravenous administration) have been confirmed in a preliminary experiment 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.
Four mL pooled blank plasma (approx. 2 mL per sex) were obtained from spare animals.

Toxicokinetic evaluation of plasma data 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 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 bioavialability 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 day of administration.

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: 3).
Statistics:
The test item treated group was compared to the reference item treated group (oral administration).
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:
relative bioavailability for Fe: approximately 0.96% for Fe present in the pigment
Bioaccessibility (or Bioavailability) testing results:
An absolute bioavailability of 1.87%/0.57% (m/f) calculated from soluble crystalline iron following oral administration compared to intravenous administration, and a relative bioavailability of approx. 0.96%/0.58 (m/f) for Fe 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.

 

CLINICAL SIGNS, MORTALITY, AND BODY WEIGHT

Reaction mass of fumes, silica and diiron trioxide:

- none of the rats treated with the test item showed any changes in behaviour or external appearance.

- faeces of the animals were normally formed.

- none of the rats died prematurely.

- individual body weights ranged from 218 to 248 g for the males and from 163 to 205 g for the females on test day 1 and were within the expected range.

 

Reference item (oral administration)

- none of the rats treated with the reference item showed any changes in behaviour or external appearance.

- faeces of the animals were normally formed.

- none of the rats died prematurely.

- individual body weights ranged from 218 to 248 g for the males and from 163 to 205 g for the females on test day 1 and were within the expected range.

 

Reference item (intravenous administration):

- slightly reduced motility was noted immediately after administration for all animals treated once intravenously with the reference item. Reduced motility lasted for 10 minutes.

- faeces of the animals were normally formed.

- none of the rats died prematurely.

- individual body weights ranged from 218 to 248 g for the males and from 163 to 205 g for the females on test day 1 and were within the expected range.

TEST ITEM FORMULATION ANALYSIS

Concentration of iron:

1) iron:

total found after all digestion (in supernatant): 48.71 mg

Conclusions:
In a relative bioavailability study, the relative bioavailability of orally administered pigment was calculated 0.96/0.58% Fe (m/f) in relation to a mixture of soluble Fe3+compound (Fe(C6H5O7)*H2O)injected i.v..
Executive summary:

  

In conclusion, the oral relative bioavailability of the pigment "Reaction mass of fumes, silica and diiron trioxide" can be assumed to be negligible.

The relative bioavailability of orally administered pigment was calculated 0.96/0.58% Fe (m/f) in relation to a mixture of soluble Fe3+compound (Fe(C6H5O7)*H2O)injected i.v..

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 element Fe contained in the pigment.


(1)  In in-vitro dissolution experiments in five different artificial physiological media after 24h, dissolved Fe and Si concentrations from this pigment were below 63 μg/L and 413μg/L, respectively, even at the highest loading of 0.1g/L, corresponding to a solubility of less than 0.5%,


(2)  In a 28-day oral toxicity study with 1,000 mg/kg pigment minimal or no increase in Fe 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 << 0.005 % (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, 110.7% Fe, were excreted via faeces within 3 days, with only <0.016% of the dose being excreted via urine at the same time.


(4)  In a relative bioavailability study, the relative bioavailability of orally administered pigment was calculated 0.95/0.58% Fe (m/f) in relation to a mixture of soluble Fe3+compound (Fe(C6H5O7)*H2O)injected i.v..


 


Comparing the findings of in-vitro dissolution testing (1) with in-vivo results (2-4), the in-vivo data consistently demonstrates slightly lower bioavailability. This is in agreement with the general understanding that in-vitro experiments in simulated gastric juice provide a conservative estimate of actual (in-vivo) bioavailability.


In conclusion, the oral relative bioavailability of the pigment “High-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix” can be assumed to be negligible, as demonstrated in three independent in-vivo studies in rats yielding very comparably results supported by an in-vitro dissolution 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 and (ii) a mass balance study involving a single dose of 1,000 mg pigment/kg bw (0.016% for Fe).


 


Absorption rate - oral: 0.01 %

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 “High-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix” 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 of in-vitrobioaccessibility experiments (Herting, Wallinder, 2010)


The chemical and physiological properties of the pigment 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 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 relevantexposure 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 silicon from the test item was in a range of below L.O.D. (pH 4.5) and 96.6 μg/L (pH 7.4) at a loading of 0.1g/L after 2 hours and in a range of below L.O.D. (pH 6.5) and 413 μg/L (pH 7.2) after 24 hours. Further, the dissolution of iron from the test item was in a range of 4.8 (pH 7.4) and 13.4 μg/L (pH 6.5) after 2 hours and in a range of 5.3 (pH 6.5) and 62.6 μg/L (pH 1.7) after 24 hours.


 


In conclusion, since the dissolved Si and Fe concentrations from this pigment were below 430 μg/L even at the highest loading of 0.1g/L, corresponding to a solubility of less than 0.45 %, this pigment may reasonably be considered biologically inert.


 


Toxicokinetic screening data from a 28 day repeated dose oral toxicity study (Leuschner, 2018)


In a 28 day repeated dose toxicity study, male and female rats were given a daily dose of the pigment "High-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix" 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 silicon and iron content.


 


The uptake of iron during a 24 hour urine and plasma sampling period was demonstrated to be negligible considering that <<0.005% of the dose was excreted via urine, mirrored by either minimal or no increases in blood plasma concentrations.


 


The iron concentrations of the 24h-urine samples, collected during the day before final sacrifice, ranged from: 51.0 - 68.8μg/L urine (mean: 58.2 ± 6.66) and 34.6 - 101μg/L urine (mean: 68.0 ± 31.1),for the male and female animals of the control group, respectively.


 


For the dosed group, the concentrations were 32.8 - 57.3μg/L urine (mean: 47.6 ± 9.56) and 50.7 - 246μg/L urine (mean: 122 ± 78.5)for the male and female animals of the dose group, respectively.


 


Following a subtraction of the background urinary element excretion (control group), and taking into account the excreted urine volume (mean 21.0 mL (m) and 16.3 mL (f)) and the body weight of the animals at the end of the study (mean 378 g (m) and 212.5 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 << 0.001 % (m) or 0.0043% (f) were found in the terminal 24-h urine collection period.


 


Summary of comparative Mass-Balance Study (Leuschner 2018a):


In a comparative mass balance study involving oral dosing of (i) the inorganic pigment "High-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix" and (ii) soluble salts of the element contained therein (Fe3+), 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 or 444 mg Fe(C6H5O7)*H2O (corresponding to 94.31 mg/kg Fe). 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 aluminium and manganese. 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 94.31 mg Fe/Kg bw (administered asFe(C6H5O7)*H2O) excreted 128.3 % (Fe), of the administered dose (as mean, male and female animals) via urine and faeces during the first three days after exposure.


 


The largest fraction (110 % Fe) was excreted via faeces and urine (<0.1% Fe) already within the first 24h.


Animals that received 1000 mg pigment /kg bw excreted 110.7% Fe 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 98.1% of Fe were excreted via faeces as largest fraction. Further 11.1% and 1.5% (Fe), were excreted via faeces on the second and third day.


Urinary excretion was negligible and below 0.016% Fe.


The mass balances for Fe is essentially complete and indicatess that the element Fe contained in the pigment "High-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix", present as Fe3+, is not absorbed in the gastrointestinal tracts to any significant extent, but pass the animal effectively unchanged. 


 


Summary of relative bioavailability study (Leuschner 2018b):


A relative bioavailability study involving serum kinetics over a period of 72 hours p. a. involving an i. v. dosing of a soluble Fe reference substances (iron citrate mono hydrate) compared to single oral dose of the same substance 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) 4.5 mg/kg bw Fe(C6H5O7)*H2O intravenously, (2) and 444 mg/kg bw Fe(C6H5O7)*H2O 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 Fe.


Cmax-levels in plasma of 10.3 µg Fe/g and 32.7 µg Fe/g, were noted ~1 or 4 hours (tmaxas mean m/f) after intravenous administration of 4.5 mg/kg Fe(C6H5O7)*H2O for the male and female rats on test day 1, respectively.


Furthermore, Cmax-levels of 31.2 µg Fe/g and 5.0 µg Fe/g were noted ~4, or 29 hours (tmaxas mean m/f) after oral administration of 444 mg/kg Fe(C6H5O7)*H2O for the male and female rats on test day 1, respectively.


Lastly, Cmax-levels of 3.63 µg Fe/g and 8.53 µg Fe/g, were noted ~20 and 25 hours (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 iron in plasma taken before exposure at t=0 h was 3.07 µg Fe/g plasma.


The plasma concentrations declined post dosing with an elimination half-life ranging from 27 (m) to 3 (f) hours for Fe.


For Fe a relative bioavailability of 1.86/1.40% (m/f) calculated from soluble Fe(C6H5O7)*H2O following oral administration compared to intravenous administration, and of approximately 0.95/0.58% (m/f) for Fe present in the pigment.


In sum, experimentally determined plasma kinetic values for Fe are very consistent and the very low relative bioavailabilities of Fe from the pigment demonstrate 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 element Fe contained in the pigment.


 


(1)  In in-vitro dissolution experiments in five different artificial physiological media, dissolved Fe and Si concentrations from this pigment were below 63 μg/L and 420μg/L even at the highest loading of 0.1g/L, corresponding to a solubility of less than 0.5%,


 


(2)   In a 28-day oral toxicity study with 1,000 mg/kg pigment minimal or no increase in Fe 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 << 0.005 % (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, 110.7% Fe, were excreted via faeces within 3 days, with only <0.016% of the dose being excreted via urine at the same time.


 


(4)  In a relative bioavailability study, the relative bioavailability of orally administered pigment was calculated 0.95/0.58% Fe (m/f) in relation to a mixture of soluble Fe3+compound (Fe(C6H5O7)*H2O)injected i.v..


 


Comparing the findings of in-vitro dissolution testing (1) with in-vivo results (2 -4), the in-vivo data consistently demonstrates slightly lower bioavailability. This is in agreement with the general understanding that in-vitro experiments in simulated gastric juice provide a conservative estimate of actual (in-vivo) bioavailability.


 


In conclusion, the oral relative bioavailability of the pigment "High-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix" 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 and (ii) a mass balance study involving a single dose of 1,000 mg pigment/kg bw (0.016% for Fe).


 


Absorption rate - oral: 0.01 %