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Endpoint:
basic toxicokinetics in vitro / ex vivo
Remarks:
Bioaccessibility - transformation/dissolution in artificial physiological media
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Objective of study:
other: Bioaccessibility
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Series on Testing and Assessment No. 29 (23-Jul-2001): Guidance document on transformation/dissolution of metals and metal compounds in aqueous media
Deviations:
yes
Remarks:
Bioaccessibility testing: loading of 100 mg/L; five artificial physiological media agitated at 100 rpm, at 37 °C ± 2 °C; sampling after 2h and 24h; determination of Mn and Al concentrations after filtration by AAS-GF.
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 exposure:
The test item was exposed to five different test media at a pH range from 1.7 to 7.4. The following synthetic biological fluids were used:
• Gamble’s solution (GMB, pH 7.4) which mimics the interstitial fluid within the deep lung under normal health conditions (de Meringo et. Al. 1994).
• Phosphate buffered saline (PBS, pH 7.2), which is a standard physiological solution that mimics the ionic strength of human blood serum. It is widely used in the research (e.g. Norlin et al, 2002) and medical health care community (e.g. Hanawa et al, 2004, Okazaki and Gotoh, 2005) as a reference test solution for comparison of data under simulated physiological conditions.
• Artificial sweat (ASW, pH 6.5) which simulates the hypoosmolar fluid, linked to hyponatraemia (loss of Na+ from blood), which is excreted from the body upon sweating. The fluid is recommended in the available standard for testing of nickel release from nickel containing products (EN 1811, 1998).
• Artificial lysosomal fluid (ALF, pH 4.5), which simulates intracellular conditions in lung cells occurring in conjunction with phagocytosis and represents relatively harsh conditions (Moss 1979).
• Artificial gastric fluid (GST, pH 1.7), which mimics the very harsh digestion milieu of high acidity in the stomach (Hamel et al, 1998; ASTM, 2003).

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

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

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

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

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

After exposure, the samples were allowed to cool to ambient room temperature before the final pH of the test solution was measured. The test medium was then separated from the powder particles by centrifugation at 10000 rpm for 10 minutes, resulting in a visually clear supernatant with remaining particles in the bottom of the centrifuging tube. Dynamic light scattering, (Malvern Zetasizer nano ZS instrument) was used to confirm the successful removal of all pigment particles. The supernatant solution was decanted into a polypropylene storage flask and acidified to a pH less than 2 (not needed in the case of artificial gastric fluid) with 65 % pure HNO3 prior to solution analysis
Type:
other: bioaccessibility
Results:
Highest dissolution of Mn at a loading of 0.1 g/L in GST: 126 µg/L after 2 hours. Highest dissolution of Al at a loading of 0.1 g/Lin GST: 115 µg/L after 2 hours.

BET-analysis:

The specific surface area, measured by BET-analysis is 2.51 m²/g. It should be underlined that this specific surface area is measured by nitrogen absorption and includes also the surface of surface pores.

Abbreviations:

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

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

(test item)

Exposure

GMB

PBS

ASW

ALF

GST  

Material

period

pH 7.4

pH 7.2

pH 6.5

pH 4.5

pH 1.7

MnAl

2 h

27.8 ± 6.3

19.9 ± 3.1

53.3 ± 25.4

154 ± 2.3

126 ± 12.7

Mn release

24 h

7.6 ± 1.6

7.7 ± 0.9

57.7 ± 0.9

819 ± 64.7

260 ± 52.7

MnAl

2 h

19.6 ± 7.7

17.7 ± 4.6

41.7 ± 17.3

52.4 ± 2.4

115 ± 27.2

Al release

24 h

20.7 ± 6.8

13.8 ± 0.4

36.7 ± 8.9

229 ± 43.6

182 ± 19.5

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

MnAl

2 h

0.0057 ± 0.0013

0.0042 ± 0.0007

0.0110 ± 0.0048

0.031 ± 0.0001

0.010 ± 0.0011

Mn release

24 h

0.0013 ± 0.00003

0.0001 ± 0.00001

0.0010 ± 0.00002

0.014 ± 0.0008

0.0017 ± 0.0004

MnAl

2 h

0.0040 ± 0.0016

0.0037 ± 0.0009

0.0083 ± 0.0033

0.011 ± 0.0004

0.0091 ± 0.0021

Al release

24 h

0.0004 ± 0.0001

0.0002 ± 0.00001

0.0006 ± 0.0002

0.0039 ± 0.0006

0.0012 ± 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

MnAl

2 h

0.0003 ± 0.0001

0.0002 ± 0.00003

0.0005 ± 0.0002

0.0016 ± 0.00001

0.0005 ± 0.00005

Mn release

24 h

0.0001 ± 0.00002

0.0001 ± 0.00001

0.0006 ± 0.00001

0.0084 ± 0.0005

0.0010 ± 0.0002

MnAl

2 h

0.0002 ± 0.0001

0.00019 ± 0.00005

0.0004 ± 0.0002

0.0005 ± 0.00002

0.0005 ± 0.0001

Al release

24 h

0.0002 ± 0.0001

0.0001 ± 0.00001

0.0004 ± 0.0001

0.0024 ± 0.0004

0.0007 ± 0.0001

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

MnAl

2 h

0.029 ± 0.0067

0.021 ± 0.0033

0.053 ± 0.0024

0.16 ± 0.001

0.050 ± 0.0053

Mn release

24 h

0.0079 ± 0.0019

0.0079 ± 0.0008

0.060 ± 0.0013

0.84 ± 0.049

0.10 ± 0.023

MnAl

2 h

0.020 ± 0.0080

0.019 ± 0.0048

0.042 ± 0.016

0.054 ± 0.0020

0.046 ± 0.011

Al release

24 h 0.022 ± 0.0076 0.014 ± 0.001 0.038 ± 0.0098 0.24 ± 0.037 0.072 ± 0.0073

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

MnAl

24 h

0.030 ± 0.009

0.022 ± 0.002

0.098 ± 0.011

1.08 ± 0.086

0.17 ± 0.030

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

MnAl

2 h

0.52 ± 0.12

0.38 ± 0.059

0.96 ± 0.44

2.9 ± 0.013

0.91 ± 0.096

Mn release

24 h

0.14 ± 0.035

0.14 ± 0.014

1.1 ± 0.023

15.3 ± 0.88

1.9 ± 0.41

MnAl

2 h

0.043 ± 0.017

0.040 ± 0.010

0.089 ± 0.035

0.11 ± 0.0043

0.098 ± 0.023

 Al release  24 h  0.046 ± 0.016  0.030 ± 0.0017  0.081 ± 0.0021  0.50 ±0.079  0.15 ± 0.016
Conclusions:
As dissolved Mn and Al concentrations were below 819 µg/L and 229 µg/L, respectively, even at the highest loading of 0.1 g/L, referring to a solubility of 0.82 % and 0.23 %, respectively, the pigment is considered biologically inert.
Executive summary:

The chemical and physiological properties of the pigment manganese alumina pink corundum 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 manganese from the test item manganese alumina pink corundum was in a range of 19.9 (pH 7.2) and 154 μg/L (pH 4.5) after 2 hours and below 820 μg/L at a loading of 0.1 g/L after 24 hours. Further, the dissolution of aluminium from the test item manganese alumina pink corundum was in a range of 17.7 (pH 7.2) and 115 μg/L (pH 1.7) after 2 hours and below 230 μg/L (pH 4.5) at a loading of 0.1 g/L after 24 hours.

 In conclusion, since the dissolved Mn and Al concentrations from this pigment were below 820 μg/L and 230μg/L even at the highest loading of 0.1g/L, corresponding to a solubility of less than 0.82 %, this pigment may reasonably be considered biologically inert

 

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-07-06 to 2015-07-09
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 dosing: males: 53 days; females: 67 days
- Weight at dosing: males: 255.9 - 286.8 g; females: 238.4 - 264.3 g
- Housing: 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: 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: manganese alumina pink corundum: oral (gavage); reference item (mixture of MnSO4 and Al2(SO4)3): oral (gavage) and intravenously injected
Vehicle:
other: manganese alumina pink corundum: 0.8 % aqueous hydroxyl propyl methylcellulose gel; reference item (mixture of MnSO4 and Al2(SO4)3): water (oral administration) or 0.9 % NaCl solution (intravenous administration)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
1) Manganese alumina pink corundum
The test item was suspended in the vehicle to the appropriate concentration freshly on the administration day.

2) Reference item (mixture of manganese sulfate (purity: >99%) and aluminium sulfate (purity: 99.99%))
The components of the reference item were combined as follows:
a) oral administration:
manganese sulfate: 154 mg/kg bw
aluminium sulfate: 2968 mg/kg bw
b) intravenous administration
manganese sulfate: 0.15 mg/kg bw
aluminium sulfate: 3 mg/kg bw

The mixture was dissolved in the respective vehicle for oral or intravenous injection to the appropriate concentration freshly on the administration day.

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 / 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 have been selected after consultation with the Sponsor based on available toxicity data:
The oral LD50 values for aluminium and manganese sulfate are as follows:
1) aluminium sulfate: 6200 mg/kg bw
2) manganese sulfate: >2000 mg/kg bw
Furthermore, the oral bioavailabilities of soluble Al and Mn sub-stances are given in the public domain with <1% and approx. 13%, respectively.

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 Manganese alumina pink corundum/kg bw equates tothe following doses:
1) Al: 468 mg/kg bw (corresponding to 2968 mg Al2(SO4)3/kg b.w.)
2) Mn: 56 mg/kg bw (corresponding to 154 mg MnSO4/kg bw)
The dosage administered by intravenous injection was set to 0.1% 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 as follows:
Al: 0.468 mg/kg bw (corresponding to 3 mg aluminium sulfate/kg bw)
Mn: 0.056 mg/kg bw (corresponding to 0.15 mg manganese sulfate/kg bw)
The dose levels for the reference item (oral and intravenous) were confirmed in two preliminary experiments:

1) First preliminary experiment:
Both animals treated once orally with a mixture composed of 154 mg manganese sulfate and 2968 mg aluminium sulfate/kg bw revealed slight pilo-erection starting approx. 15 minutes p.a. lasting for 4 hours.
Both animals treated once intravenously with a mixture composed of 15 mg manganese sulfate and 297 mg aluminium sulfate/kg bw died during the i.v. injection.

2) Second preliminary experiment:
The male animal treated once intravenously with 27.5 mg manganese sulfate/kg bw revealed convulsions and a lateral position immediately p.a. and died 2 minutes p.a.
The female animal treated once intravenously with 27.5 mg manganese sulfate/kg bw revealed convulsions and ataxia 2 minutes p.a., followed by moderate pilo-erection and severely reduced motility up to 20 minutes p.a. Subsequently, slight pilo-erection and slightly reduced motility were observed.
The male and the female animal treated once intravenously with 63.6 mg aluminium sulfate/kg bw revealed convulsions, a lateral position and gasping breathing immediately p.a. and died 2 minutes p.a.
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 isolated plasma was frozen at -20°C or colder, and stored at this temperature until analysis.
2 mL pooled blank plasma per sex (2 x 2 mL) were obtained from spare animals.

A pharmacokinetic evaluation of the plasma data will be performed. A non-compartment model will be employed. The following parameters will be determined, if possible:
AUC0-∞ = extrapolated area under the curve from zero to infinity
AUC0-t last = extrapolated area under the curve from time zero to the last quantifiable plasma concentration > LOQ
Kel = elimination rate constant
t1/2 = elimination half-life

Cmax values are the highest measured plasma concentrations and tmax values are the time-points of the highest plasma concentrations.

Elimination rate constants (Kel) and plasma elimination half-lives (t1/2) 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 terminal elimination rate constant. Plasma concentrations at time zero will be taken to be those at the first blood sampling time.

Furthermore, the 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 calculation.

The bioavailability of the reference item will be calculated using the following equation:
Bioavailability = (AUC p.o. dose i.v.)/(AUC i.v. dose p.o) x 100%

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 (approx. 5 mL) of each test and reference item that was mixed with a vehicle were stored at ≤ -20°C until analysis (Number of samples: 3).
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.
Type:
absorption
Results:
Cmax (plasma) of 1.97 μg Al/g & 2.38 μg Al/g & 0.11 μg Mn/g & 0.07 μg Mn/g were noted to 1.8 to 12.6 h (tmax as range of both analytes m/f) after i.v. administration of a mixture of 0.15 mg MnSO4 & 3 mg Al2(SO4)3/kg bw, for the m & f rats on day 1, resp.
Type:
absorption
Results:
Cmax-levels of 0.74 μg Al/g & 0.73 μg Al/g & 1.11 μg Mn/g & 1.38 μg Mn/g were noted 1.0 to 4.2 hours (tmax as range of both analytes m/f) after oral administration of a mixture of 154 mg MnSO4 & 2968 mg Al2(SO4)3/kg bw, for the m & f rats on day 1, resp.
Type:
absorption
Results:
Cmax-levels of 1.13 μg Al/g and 0.69 μg Al/g and 0.03 μg Mn/g and 0.03 μg Mn/g were noted 0.8 to 19.8 hours (tmax as range of both analytes m/f) after oral administration of 1000 mg pigment/kg for the male and female rats on test day 1, respectively.
Type:
excretion
Results:
The plasma concentrations declined post dosing with an elimination half-life ranging from 2.3 to 13 hours for Al and from 1.6 to 10.8 hours for Mn.
Details on absorption:
Cmax-levels in plasma of 1.97 μg Al/g and 2.38 μg Al/g and 0.11 μg Mn/g and 0.07 μg Mn/g were noted to 1.8 to 12.6 hours (tmax as range of both analytes m/f) after intravenous administration of a mixture of 0.15 mg MnSO4 and 3 mg Al2(SO4)3/kg b.w., for the male and female rats on test day 1, respectively.

Furthermore, Cmax-levels of 0.74 μg Al/g and 0.73 μg Al/g and 1.11 μg Mn/g and 1.38 μg Mn/g were noted 1.0 to 4.2 hours (tmax as range of both analytes m/f) after oral administration of a mixture of 154 mg MnSO4 and 2968 mg Al2(SO4)3/kg b.w., for the male and female rats on test day 1, respectively.

Lastly, Cmax-levels of 1.13 μg Al/g and 0.69 μg Al/g and 0.03 μg Mn/g and 0.03 μg Mn/g were noted 0.8 to 19.8 hours (tmax as range of both analytes 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 aluminium in plasma taken before exposure at t=0 h was 0.66 μg Al/g and for manganese the mean blank plasma concentration was 0.01 μg Mn/g plasma.

Please also refer to the field "Attached background material" below.
Details on distribution in tissues:
no data
Details on excretion:
The plasma concentrations declined post dosing with an elimination half-life ranging from 2.3 to 13 hours for Al and from 1.6 to 10.8 hours for Mn.

Please also refer to the field "Attached background material" below.
Toxicokinetic parameters:
other: bioavailability
Remarks:
relative bioavailability: 0.11/0.008% (m/f) for Al present in the pigment. Relative bioavailability 0.02/0.12% (m/f) for Mn present in the pigment.
Metabolites identified:
not specified
Details on metabolites:
no data
Bioaccessibility (or Bioavailability) testing results:
For Al an absolute bioavailability of 0.10/0.05 % (m/f) calculated from soluble Al2(SO4)3 following oral administration compared to intravenous administration, and a relative bioavailability of approximately 0.11/0.08% (m/f) for Al present in the pigment.

An absolute bioavailability of 0.18/2.38% (m/f) was calculated for Mn from soluble MnSO4 following oral administration compared to intravenous administration, and a relative bioavailability of approximately 0.02/0.12% (m/f) for Mn present in the pigment.

Please also refer to the field "Attached background material" below.

LOCAL TOLERANCE (REFERENCE ITEM; INTRAVENOUS ADMINISTRATION)

No signs of local intolerance reactions were observed for the male or female rats treated once intravenously with the reference item.

CLINICAL SIGNS AND MORTALITY

Manganese alumina pink corundum:

- slightly reduced motility was noted for the animals treated with the test item starting 30 minutes p.a. and lasting for 30 minutes.

- faeces of all animals were of normal consistency throughout the experimental period.

- none of the animals died prematurely.

Reference item (oral administration):

- slightly reduced motility was noted for the animals treated with the reference item starting 30 minutes p.a. and lasting for 30 minutes.

- faeces of all animals were of normal consistency throughout the experimental period.

- none of the animals died prematurely.

 

Reference item (intravenous administration):

- no changes in behaviour and external appearance were noted during the course of the study.

- faeces of all animals were of normal consistency throughout the experimental period.

- none of the animals died prematurely.

 

BODY WEIGHT

The body weight of the male and female animals treated either with manganese alumina pink corundum (oral administration) or the reference item (oral or intravenous administration) ranged from 255.9 – 286.8 g for the males and from 238.4 – 264.3 g for the females on test day 1.

TEST ITEM FORMULATION ANALYSIS

Concentration of aluminium and manganese in application solution:

1) aluminium:

Nominal concentration: 593 mg

Actual concentration: 570 mg

Recovery [%]: 96.1 %

2) manganese:

Nominal concentration: 70.05 mg

Actual concentration: 64.5 mg

Recovery [%]: 92.0 %

Conclusions:
In a relative bioavailability study, the relative bioavailability of orally administered pigment was calculated 0.11% (Al) and 0.12% (Mn) in relation to a mixture of soluble Al3+and Mn2+ compounds (Al2(SO4)3, MnSO4) injected i.v..
Executive summary:

In conclusion, the oral relative bioavailability of the pigment "Manganese alumina pink corundum" can be assumed to be negligible.  The relative bioavailability of orally administered pigment was calculated 0.11% (Al) and 0.12% (Mn) in relation to a mixture of soluble Al3+and Mn2+compounds (Al2(SO4)3, MnSO4)injected i.v..

Endpoint:
basic toxicokinetics, other
Remarks:
mass balance
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-01-23 to 2015-01-26
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 manganese alumina pink corundum, vehicle (0.8 % aqueous hydroxyl propyl methylcellulose gel), or reference item (mixure composed of aluminium sulfate and manganese sulfate) 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: 49 days; females: 53 days
- Weight at dosing: males: 238 - 260 g; females: 182 - 209 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: manganese alumina pink corundum: 0.8 % aqueous hydroxyl propyl methylcellulose gel; reference item (mixture of MnSO4 and Al2(SO4)3): water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
1) Manganese alumina pink corundum:
The test item formulation was freshly prepared on the administration day by dissolving the test item in the vehicle to the appropriate concentration. 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 and reference item was adjusted to the animal's current body weight on the administration day.

2) Reference item (mixture of manganese sulfate (assay: 100 %) and aluminium sulfate (assay: 54.6 %))
The components of the reference item were combined as follows:
aluminium sulfate: 2968 mg/kg bw
manganese sulfate: 154 mg/kg bw
The reference item formulation was freshly prepared on the administration day by dissolving the reference item in the vehicle to the appropriate concentration. 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 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:
yes, concurrent vehicle
Positive control reference chemical:
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 values for the reference items were as follows:
1) aluminium sulfate: >2000 - 6200 mg/kg bw
2) manganese sulfate: >2000 mg/kg bw
Furthermore, oral bioavailabilities of soluble Al and Mn substances are given in the public domain as <1% and approx. 13%, respectively.

The oral test item dose of 1000 mg/kg bw corresponds to the limit dose used in a separate 28-day oral toxicity study, which is considered to be the maximum feasible dose. Based on the chemical composition of the test item, a dose of 1000 mg manganese alumina pink corundum/kg bw equates to the following doses:
1) Al: 468 mg/kg bw (corresponding to 2968 mg aluminium sulfate/kg bw)
2) Mn: 56 mg/kg bw (corresponding to 154 mg manganese sulfate/kg bw)

The dose level for the reference item was confirmed in a preliminary experiment employing two animals (please also refere to IUCLID Section 7.1.1: k_Leuschner_2017_ in vivo). In this preliminary experiment, both animals were treated once orally with a mixture composed of 2968 mg Al2(SO4)3 and 154 mg MnSO4/kg bw and revealed slight pilo-erection starting approx. 15 minutes p.a. lasting for 4 hours.
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 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, intraabdominal 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 was 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.
Type:
absorption
Results:
Absorption rate - oral: 0.01 %
Details on absorption:
Urinary excretion for all elements was negligible and below 0.005% for Mn, and <0.001% for Al.
Absorption rate - oral: 0.01 %
Details on excretion:
Animals that received 1000 mg pigment /kg bw excreted 95.5% Al and 144% Mn 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 93.7% of Al, and 109.6% of Mn were excreted via faeces as largest fraction. Further 0.3% and 1.5% (Al), and 16.7% and 17.9% (Mn) were excreted via faeces on the second and third day. Urinary excretion for all elements was negligible and below 0.005% for Mn, and <0.001% for Al.

CLINICAL SIGNS, MORTALITY, BODY WEIGHT, GROSS PATHOLOGY

Vehicle control group:

- no systemic intolerance was observed

- faeces of control animals were normally formed.

- none of the rats died prematurely.

- individual body weights ranged from 239.4 to 260.2 g for the males and from 155.1 g to 163.0 g for the females on test day 1 and were within the expected range.

- no influence was noted on the body weight at autopsy.

- no pathological findings were recorded

Manganese alumina pink corundum:

- 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 238.8 to 255.5 g for the males and from 154.4 g to 165.8 g for the females on test day 1 and were within the expected range.

- no influence was noted on the body weight at autopsy.

- 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 animals treated with the test item.

 

Reference item:

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

- none of the rats died prematurely.

- individual body weights ranged from 244.4 to 260.2 g for the males and from 156.0 g to 164.4 g for the females on test day 1 and were within the expected range.

- no influence was noted on the body weight at autopsy.

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

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

- statistically significant differences in organ weights compared to vehicle control group which are not considered to be reference item-related are as follows:

females (test day 4): decreased relative spleen weight, decreased absolute pituitary, and decreased absolute spleen weight (p ≤ 0.05).

Conclusions:
Animals that received 1000 mg pigment /kg bw excreted 95.5% Al and 144% Mn 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 93.7% of Al, and 109.6% of Mn were excreted via faeces as largest fraction. Further 0.3% and 1.5% (Al), and 16.7% and 17.9% (Mn) were excreted via faeces on the second and third day.

Urinary excretion for all elements was negligible and below 0.005% for Mn, and <0.001% for Al.

Manganese 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 Mn(II) of about 1.57 mg/animal (average of 20 animals, male and female) seems reasonable which increases the total dose by 12.5%. Ten per cent diet uptake per animal/day may be underestimated so that the obtained results are considered to be realistic.

Only ca. 4.5% of Al remains unaccounted. It may be considered that delayed excretion of the whole dose is reasonable since at the 72h sampling still ~1.5% of the Al dose (mean, mainly female animals) was excreted. Further, it may be considered that the actually received dose did not fully correspond to the nominal dose as calculated. These aspects were not further addressed within the context of this study.
In total, the mass balances for manganese and aluminium is essentially complete and indicates that the elements contained in the pigment " Manganese alumina pink corundum", present as Al(III) and Mn(II), are not absorbed in the gastrointestinal tracts to any significant extent, but pass the animal effectively unchanged. It is not clear whether the determination of < 0.001% Mn in urine during the first day of exposure is a result of manganese absorbed from the pigment and is presumably caused by bioavailable manganese from the diet.
Executive summary:

Animals that received 1000 mg pigment /kg bw excreted 95.5% Al and 144% Mn 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 93.7% of Al, and 109.6% of Mn were excreted via faeces as largest fraction. Further 0.3% and 1.5% (Al), and 16.7% and 17.9% (Mn) were excreted via faeces on the second and third day.

Urinary excretion for all elements was negligible and below 0.005% for Mn, and <0.001% for Al.

The mass balances for Al and Mn are essentially complete and indicate that the elements contained in the pigment "Manganese alumina pink corundum", present as Al3+and Mn2+, are not absorbed in the gastrointestinal tracts to any significant extent, but pass the animal effectively unchanged. 

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 Al and Mn contained in the pigment.

(1)   In in-vitro dissolution experiments in five different artificial physiological media, dissolved Mn and Al concentrations from this pigment were below 820 μg/L and 230 μg/L even at the highest loading of 0.1 g/L, corresponding to a solubility of less than 0.82 %,

(2)   In a 28-day oral toxicity study with 1,000 mg/kg pigment no increase in Al and Mn 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 < 0.002 % (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, 95.5 % Al, and 144 % Mn of the dose were excreted via urine and faeces within 3 days, with only <0.005 % of the dose being excreted via urine.

(4)   In a relative bioavailability study, the relative bioavailability of orally administered pigment was calculated 0.09 % (Al) and 0.07 % (Mn) in relation to a mixture of soluble Al3+ and Mn2+ compounds (Al2(SO4)3, MnSO4) 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 "Manganese alumina pink corundum" 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 (<<0.002 % for both metals) and (ii) a mass balance study involving a single dose of 1,000 mg pigment/kg bw (0.004 % for Mn and 0.0002 % for Al).

 

Absorption rate - oral: 0.01 %

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
0.01

Additional information

The toxicity data in this registration dossier refer explicitly to the pigment Manganese alumina pink corundum 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-vitro bioaccessibility experiments (Herting, Wallinder, 2010)


The chemical and physiological properties of the pigment manganese alumina pink corundum 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 manganese from the test item manganese alumina pink corundum was in a range of 19.9 (pH 7.2) and 154 μg/L (pH 4.5) after 2 hours and below 820 μg/L at a loading of 0.1 g/L after 24 hours. Further, the dissolution of aluminium from the test item manganese alumina pink corundum was in a range of 17.7 (pH 7.2) and 115 μg/L (pH 1.7) after 2 hours and below 230 μg/L (pH 4.5) at a loading of 0.1g/L after 24 hours.


 


 In conclusion, since the dissolved Mn and Al concentrations from this pigment were below 820 μg/L and 230 μg/L even at the highest loading of 0.1g/L, corresponding to a solubility of less than 0.82 %, 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 "Manganese alumina pink corundum" 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 manganese and aluminium content.


The uptake of manganese and aluminium during a 24 hour urine and plasma sampling period was demonstrated to be negligible considering that <<0.002 % of the dose was excreted via urine for both metals, mirrored by either minimal or no increases in blood plasma concentrations.


The manganese and aluminium concentrations of the 24h-urine samples, collected during the day before final sacrifice, ranged from: 2.43 - 10.0μg/L urine(mean: 6.33 ± 3.70) and 5.02 - 38.7 μg/L urine(mean: 14.7 ± 13.8) for Mn and from < LOD - 6.72 μg/L urine (mean: 4.13 ± 2.27) and < LOQ - 11.1 μg/L urine (mean: 8.42 ± 2.47) for Al for the male and female animals of the control group, respectively.


For the dosed group, the concentrations were 5.48 - 69.6μg/L urine (mean: 29.8 ± 26.3) and 6.78 - 18.7μg/L urine (mean: 11.6 ± 4.89) for Mn and 7.39 - 172 μg/L urine (mean: 52.1 ± 79.8) and 1.66 - 46.5 μg/L urine (mean: 26.0 ± 16.4) for Al for the male and female animals, respectively.


Following a subtraction of the background urinary element excretion (control group), and taking into account the excreted urine volume (mean 9.9 mL (m) and 10.3 mL (f)) and the body weight of the animals at the end of the study (mean 311.2 g (m) and 221.92 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 0.0017 % (m) or 0.0002 % (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 "Manganese alumina pink corundum" and (ii) soluble salts of the elements contained therein (Mn2+and Al3+), 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 2968 mg Al2(SO4)3, and 154 mg MnSO4/kg(corresponding to 468.9 mg/kg Al, and 56.1 mg/kg Mn). 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 a mixture of 468.9 mg Al/Kg bw (administered as Al2(SO4)3and 56.1 mg Mn/Kg bw (administered as MnSO4) excreted 48.7 % (Al), and 121% (Mn) of the administered dose (as mean, male and female animals) via urine and faeces during the first three days after exposure.


The largest fraction (31.2 % for Al, and 58.9 % for Mn) was excreted via faeces and urine (0.1 % for Al and 0.25 % for Mn) already within the first 24h.


Approximately 51 % of the administered dose for Al remain unaccounted for in this group. Due to the fact that Al3+ has a very low absorbance ability within the g.i. tract (~0.9 %) a hypothesis that this fraction was contained in the exsanguinated blood of the animals or has distributed to the organs seems not appropriate. It may be considered that delayed excretion of the whole dose is reasonable since at 72h still ~5 % of the Al dose (mean, mainly female animals) was excreted.


Further it may be considered that the actually received dose did not fully correspond to the nominal dose as calculated. These aspects were not further addressed within the context of this study.


Manganese 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 Mn2+ of about 1.57 mg/animal (average of 20 animals, male and female) seems reasonable which increases the total dose by 12.5 %. 10 % diet uptake per animal/day may be underestimated so that the obtained results are considered to be realistic.


Animals that received 1000 mg pigment /kg bw excreted 95.5 % Al and 144 % Mn 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 93.7 % of Al, and 109.6 % of Mn were excreted via faeces as largest fraction. Further 0.3 % and 1.5 % (Al), and 16.7 % and 17.9 % (Mn) were excreted via faeces on the second and third day.


Urinary excretion for all elements was negligible and below 0.005 % for Mn, and <0.001 % for Al.


Considering the uptake of additional Mn2+ by the diet as explained above the mass balances for Al and Mn are essentially complete and indicate that the elements contained in the pigment "Manganese alumina pink corundum", present as Al3+ and Mn2+, are 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 Al, and Mn reference substances (aluminium sulfate, manganese sulfate) 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) a mixture of 3 mg/kg Al2(SO4)3and 0.15 mg/kg bw MnSO4intravenously, (2) a mixture of 2968 mg/kg Al2(SO4)3, and 101 mg/kg bw MnSO4via 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 elements Al, and Mn.


 


Cmax-levels in plasma of 1.97 µg Al/g and 2.38 µg Al/g, and 0.11 µg Mn/g and 0.07 µg Mn/g were noted ~2 or 12.6 hours (tmaxas mean m/f) after intravenous administration of a mixture of 3 mg/kg Al2(SO4)3and 0.15 mg/kg bw MnSO4for the male and female rats on test day 1, respectively.


Furthermore, Cmax-levels of 0.74 µg Al/g and 0.73 µg Al/g and 1.11 µg Mn/g and 1.38 µg Mn/g were noted 1.0, or 4.2 hours (tmaxas mean m/f) after oral administration of a mixture of 2968 mg/kg Al2(SO4)3, and 154 mg/kg bw MnSO4for the male and female rats on test day 1, respectively.


Lastly, Cmax-levels of 1.13 µg Al/g and 0.69 µg Al/g, and 0.03 µg Mn/g and 0.03 µg Mn/g were noted 0.8 and 19.8 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 aluminium and manganese in plasma taken before exposure at t=0 h was 0.66 µg Al/g and for manganese the mean blank plasma concentration was 0.01 µg Mn/g plasma.


The plasma concentrations declined post dosing with an elimination half-life ranging from 2.3 to 13.0 hours for Al and from 1.6 to 10.8 hours for Mn.


 


For Al a relative bioavailability of 0.101/0.051% (m/f) calculated from soluble Al2(SO4)3following oral administration compared to intravenous administration, and of approximately 0.107/0.078% (m/f) for Al present in the pigment.


Relative bioavailability of 0.178/2.375% (m/f) was calculated for Mn from soluble MnSO4following oral administration compared to intravenous administration, and of approximately 0.02/0.12% (m/f) for Mn present in the pigment.


In sum, experimentally determined plasma kinetic values for Al and Mn are very consistent and the very low relative bioavailabilities of Al and Mn 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 elements Al and Mn contained in the pigment.


 


(1)   In in-vitro dissolution experiments in five different artificial physiological media, dissolved Mn and Al concentrations from this pigment were below 820 μg/L and 230 μg/L even at the highest loading of 0.1 g/L, corresponding to a solubility of less than 0.82 %,


 


(2)   In a 28-day oral toxicity study with 1,000 mg/kg pigment no increase in Al and Mn 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 < 0.002 % (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, 95.5 % Al, and 144 % Mn of the dose were excreted via urine and faeces within 3 days, with only <0.005 % 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 0.09 % (Al) and 0.07% (Mn) in relation to a mixture of soluble Al3+and Mn2+compounds (Al2(SO4)3, MnSO4)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 "Manganese alumina pink corundum" 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 (<<0.002 % for both metals) and (ii) a mass balance study involving a single dose of 1,000 mg pigment/kg bw (0.004 % for Mn and 0.0002 % for Al).


 


Absorption rate - oral: 0.01 %