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Toxicological information

Basic toxicokinetics

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Administrative data

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

Data source

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

Materials and methods

Objective of study:
other: Bioaccessibility
Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Series on Testing and Assessment No. 29 (23-Jul-2001): Guidance document on transformation/dissolution of metals and metal compounds in aqueous media
Deviations:
yes
Remarks:
Bioaccessibility testing: loading of 100 mg/L; five artificial physiological media agitated at 100 rpm, at 37 °C ± 2 °C; sampling after 2h and 24h; determination of 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

Test material

Constituent 1
Chemical structure
Reference substance name:
Manganese alumina pink corundum
EC Number:
269-061-0
EC Name:
Manganese alumina pink corundum
Cas Number:
68186-99-2
Molecular formula:
Mn(x)Al(2-x)O3 0,01≤x≤0,25
IUPAC Name:
Manganese alumina pink corundum
Test material form:
solid: particulate/powder
Details on test material:
- Chemical description: Manganese Alumina Pink Corundum
- Substance type: inorganic pigment
- Physical state: solid, pink powder, odourless, Hematite-corundum structure
- Storage condition of test material: at room temperature

Test animals

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

Administration / exposure

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

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

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

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

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

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

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

Results and discussion

Main ADME results
Type:
other: bioaccessibility
Results:
Highest dissolution 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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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