Cobalt aluminate blue spinel

Basic toxicokinetics

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

Study period:

2012-0813 to 2013-02-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Objective of study:

bioaccessibility (or bioavailability)

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 Co and Al concentrations after filtration by ICP-OES and ICP-MS.

Principles of method if other than guideline:

An internationally agreed guideline does not exist for this test (e.g. OECD). However, similar tests have been conducted with several metal compounds, including steels, in previous risk assessments (completed under Regulation (EEC) No 793/93) and in recent preparation for REACH regulation (EC) No 1907/2006.
The test was performed on the basis of the guidance for OECD-Series on testing and assessment Number 29 and according to the bioaccessibility test protocol provided by the study monitor.

GLP compliance:

yes (incl. QA statement)

Remarks:

signed, 2011-02-07

Test material

Test material information

Constituent 1

Reference substance name:

Cobalt aluminate blue spinel

EC Number:

310-193-6

EC Name:

Cobalt aluminate blue spinel

Cas Number:

1345-16-0

Molecular formula:

Co(x)Al(3-x)O4 1≤x≤1,5

IUPAC Name:

Cobalt aluminium spinel

Test material form:

solid: particulate/powder

Details on test material:

- Substance name: Cobalt aluminate blue spinel
- C.I. name: Pigment blue 28
- Substance type: inorganic pigment
- Physical state: solid, blue powder, odourless
- Storage condition: Keep dry in closed containers

Test animals

Species:

other: in vitro (simulated human body fluids)

Administration / exposure

Details on exposure:

Test principle in brief:
- five different artificial physiological media,
- single loading of test substance of 100 mg/L,
- samples taken after 2 and 24 hours agitation (100 rpm) at 37 ± 2 °C,
- two method blanks per artificial media were tested; measurement (by ICP-OES and ICP-MS) of dissolved aluminium and cobalt concentrations after filtration (0.2 µm, Supor membrane)
- the study was performed in duplicates

The aim of this test was to assess the dissolution of IPC-2013-011 (Cobalt aluminate blue spinel) in five artificial physiological media: Artificial lysosomal fluid (ALF, pH = 4.5), Artificial sweat solution (ASW, pH = 6.5), Gamble´s solution (GMB, pH = 7.4), Artificial gastric fluid (GST, pH = 1.5), Phosphate buffered saline (PBS, pH = 7.4). The test media were selected to simulate relevant human-chemical interactions (as far as practical), i.e. a substance entering the human body by ingestion into the gastrointestinal tract and by inhalation.

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:

Reagents
The water (resistivity >18 MΩ·cm.) used for this test was purified with a Pure Lab Ultra water purification system from ELGA LabWater, Celle, Germany.
- Nitric acid - “Supra” quality (ROTIPURAN® supplied by Roth, Karlsruhe, Germany).
- Hydrochloric acid – “instra-analyzed plus” quality (J.T. Baker, Griesheim, Germany).

Metal analysis
- Standards: single and multielement standards: Merck XXI (lot no. HC075495); Merck VIII (lot no. HC137073); Merck Certipur Cobalt ICP standard 1000 mg/L (lot no. HC242065); Merck Certipur Aluminium ICP standard 1000 mg/L (lot no. HC125228, Darmstadt, Germany).
- Certified reference materials: TMDA-70 (lot no. 0310, Environment Canada) and a multielement standard: Merck VIII (lot no. HC137073); Merck XXI (lot no. HC075495, Darmstadt, Germany)

Instrumental and analytical set-up for the ICP-OES instrument (Al measurement series):
Agilent 720, Agilent Technologies, Waldbronn, Germany
Nebulizer: Sea spray nebulizer from Agilent
Spray chamber: Glass cyclonic spray chamber from Agilent
Carrier gas flow: 0.75 L/min
RF power: 1200 W
Wavelengths: Aluminium: 176.502 nm, 172.438 nm, 257.509 nm, 308.215 nm and 394.401 nm
Calibration: blank, 5 µg/L, 10 µg/L, 50 µg/L, 100 µg/L, 200 µg/L and 300 µg/L
Correlation coefficients (r): at least 0.9999

The applied LOD/LOQ calculations for the Agilent 720 ICP-OES are (according to DIN 32645):
LOD: 3 * standard deviation of calibration blank/slope of the calibration;
LOQ: 3 * LOD.

Instrumental and analytical set-up for the ICP-OES instrument (mass balance series):
Thermo IRIS Intrepid II from Thermo Electron Corporation, Germany
Nebulizer: Concentric glass nebulizer, from Thermo
Spray chamber: Glass cyclonic spray chamber, from Thermo
Nebulizer gas flow: 0.68 L/min
Make-up gas flow: 0.5 L/min
RF power: 1150 W
Wavelengths: Cobalt: 228.616 nm and 230.786 nm.
Calibration: blank, 1 µg/L, 5 µg/L, 7.5 µg/L, 10 µg/L, 25 µg/L, 50 µg/L, 100 µg/L, 200 µg/L, 300 µg/L and 400 µg/L
Correlation coefficients (r): at least 0.9999
Four measurement series were performed for the determination of aluminium and cobalt concentrations in the test item samples, method blanks, mass balance samples and filter samples.

The applied LOD/LOQ calculations for the IRIS Intrepid ICP-OES are:
LOD: 3 * method standard deviation from calibration line;
LOQ: 10 * method standard deviation from calibration line.
These data were read directly from the Thermo IRIS Intrepid II ICP-OES instrument output (data calculated by internal algorithms of the instrument software).

Instrumental and analytical set-up for the ICP-MS instrument:
Agilent 7700 ICP-MS, Agilent Technologies, Waldbronn, Germany
Nebulizer: Concentric glass nebulizer, from GlassExpansion
Spray chamber: Scott Type spray chamber, from Agilent
Carrier gas flow: 0.91 L/min
Dilution/Make-up gas flow: 0.13 L/min
RF power: 1500 W
Isotopes: 90Zr, 91Zr, 103Rh (internal standard)
calibrations: blank, 0.1 µg/L, 0.25 µg/L, 0.5 µg/L, 0.75 µg/L, 1.0 µg/L, 2.5 µg/L, 5.0 µg/L, 7.5 µg/L, 10.0 µg/L, 25.0 µg/L, 50.0 µg/L, 75.0 µg/L, 100 µg/L, 250 µg/L and 500 µg/L.
Correlation factors (r) were at least 0.9997
In sum, five series of measurements were performed for the determination of total dissolved cobalt and aluminium concentrations in samples including the test vessels as well as blanks to determine background levels of elements and fortified samples.
The LOD and LOQ for zirconium were calculated using the internal instrument algorithm. This calculation is according to DIN 32645. For this the standard deviation of calibration blanks is multiplied by 3 and divided by the slope of the calibration line. In Table 10 the LODs and LOQs for cobalt and aluminium are shown.

Details on dosing and sampling:

Loading:
The nominal loading in this test was 100 mg/L. However, due to weighing uncertainties the actual loadings range from 100.422 mg/L to 101.986 mg/L in the test vessels.

Results and discussion

Main ADME results

Type:

other: Bioaccessibility

Results:

Higest dissolution at a loading of 0.1g/L after 24h in ALF: Co: 12.9 ± 0.37 µg/L, Al: 181 ± 1.54 µg/L; in GST: Co: 9.18 ± 0.08 µg/L, Al: 223 ± 4.81 µg/L

Any other information on results incl. tables

Method validation summary (ICP-OES)

validation parameter	results	Comment
Selectivity	Nearly similar data with two different wavelengths for ICP-OES method	interference observed on the wavelength 176.502 nm, for this reason wavelength 394.401 nm was used for evaluation of date -> no interference was observed on this wavelength
Linearity	applied calibration functions were linear	correlation coefficient at least 0.9999
Limit of detection	Al: 3.45 – 4.08 µg/L Co: 0.71 – 2.58 µg/L
Limit of quantification	Al: 10.3 – 12.2 µg/L Co: 2.35 – 8.60 µg/L
Method blanks	ASW <LOQ; GST: Method blanks above LOQ but at least 9.93 fold lower than concentrations in samples; ALF: Method blanks above LOQ but at least 5.63 fold lower than concentrations in samples	Elevated concentrations of Al in method blanks origin from the applied chemicals for preparation of different media
Accuracy	mean recovery for CRM TMDA-70: Al: 105 ± 1.6 % (n = 7) Co: 100 ± 2.8 % (n = 9)	high concentration range (415 µg Al/L; 285 µg Co/L)
Trueness measurement series Al in samples	mean recovery for recalibration standard : Al: 101 ± 4.5 % (n = 7)	low concentration range (100 µg/L)
Trueness measurement series Al in samples	mean recovery for recalibration standard : Al: 100 ± 1.3 % (n = 7)	mid concentration range (250 µg/L)
Trueness mass balance samples	mean recovery for recalibration standard : Co: 101 ± 1.6 % (n = 5)	low concentration range (10 µg/L)
Trueness mass balance samples	mean recovery for recalibration standard : Co: 95.5 ± 1.5 % (n = 5)	Mid low concentration range (25 µg/L)
Trueness mass balance samples	mean recovery for recalibration standard : Co: 101 ± 1.2 % (n = 4)	Mid concentration range (100 µg/L)
Trueness mass balance samples	mean recovery for recalibration standard : Co: 100 ± 0.5 % (n = 4)	High concentration range (250 µg/L)
Trueness	Fortification of samples: Al: 86.5 – 96.8 %
Accuracy	mean recovery for CRM TMDA-70: Al: 105 ± 1.6 % (n = 7) Co: 100 ± 2.8 % (n = 9)	high concentration range (415 µg Al/L; 285 µg Co/L)

Method validation summary (ICP-MS)

validation parameter	results	Comment
Selectivity	Co: similar in Helium and HiHelium mode Al: similar in Helium and HiHelium mode	appropriate gas mode were selected for interference free measurements
Linearity	applied calibration functions were linear	correlation coefficients ≥ 0.9997
Limit of detection	Co: 0.0004 – 0.45 µg/L Al: 0.32 – 0.39 µg/L
Limit of quantification	Co: 0.001 – 1.38 µg/L Al: 0.96 – 1.17 µg/L
Method blanks	Co: ASW, PBS and GST below LOD; GMB above LOQ but at least 3.4 fold lower than concentrations in samples; ALF above LOQ but at least 91 fold lower than concentrations in samples Al: Method blanks GMB and PBS above LOQ, mainly same concentrations as in samples	Elevated concentrations of Co and Al in method blanks origin from the applied chemicals for preparation of different media
Accuracy	mean recovery for CRM TMDA-70: Co: 104 ± 6.5 % (n = 20) Al: 94.0 ± 8.2 % (n = 8)	high concentration range (285 µg Co/L, 415 µg Al/L)
Trueness Co	mean recovery for recalibration standard: Co: 98.6 ± 6.4 % (n = 20)	low concentration range (10 µg/L)
Trueness Co	mean recovery for recalibration standard: Co: 100 ± 4.5 % (n = 20)	mid concentration range (100 µg/L)
Trueness Al	mean recovery for recalibration standard: Al: 87.2 ± 6.1 % (n = 8)	low concentration range (10 µg/L)
Trueness Al	mean recovery for recalibration standard: Al: 94.1 ± 5.2 % (n = 8)	mid concentration range (100 µg/L)
Trueness	Fortification of samples: Co: 91.8– 109 % Al: 87.2– 111 %
Reproducibility	mean recovery for CRM TMDA-70: Co: 104 ± 6.5 % (n = 20) Al: 94.0 ± 8.2 % (n = 8)	high concentration range (285 µg Co/L, 415 µg Al/L)

 

Concentration of aluminium in artificial media, calculated nominal aluminium concentration and dissolved amount of aluminium.

media and sample	total Al ± SD in method blanks [µg/L]	total Al ±SD in sample vessels [µg/L]	Al ± SD in sample vessels with blank subtraction [µg/L]	calculated nominal Al concentration in [µg/L]#	dissolved amount Al in artificial media [%] normalized for measured background in method blank
ALF 2h	27.9 ± 0.12	157 ± 1.30	129 ± 1.30	31225	0.41 ± <0.01
ALF 24h	24.8 ± 0.37	206 ± 1.54	181 ± 1.54	31225	0.58 ± 0.01
ASW 2h	<LOQ	15.7 ± 0.13	15.7 ± 0.13	31106	0.05 ± <0.01
ASW 24h	<LOQ	22.5 ± 1.52	22.5 ± 1.52	31106	0.07 ± 0.01
GMB 2h	37.7 ± 5.30	31.7 ± 3.49	1.51	31153	0.005
GMB 24h	27.9 ± 4.83	34.7 ± 1.52	6.80 ± 1.52	31153	0.02 ± 0.01
GST 2h	18.3 ± 0.26	182 ± 3.59	163 ± 3.59	31273	0.52 ± 0.01
GST 24h	15.3 ± 1.52	238 ± 4.81	223 ± 4.81	31273	0.71 ± 0.02
PBS 2h	14.6 ± 0.78	9.38 ± 0.75	-	31156	-
PBS 24h	10.0 ± 0.16	10.4 ± 1.35	-	31156	-

# (initial weight (e.g. 50 mg) * 30.75 § (percentage aluminium in test item) / 100) * 2 (multiplication to calculate aluminium amount in one litre -> 100 mg/L) = nominal aluminium concentration in [mg/L] / 1000 = nominal aluminium concentration in [µg/L]

§according to CoA 58.10 % Al as Al₂O₃ => 52.93 % aluminium in Al₂O₃ => (58.10 % * 52.93 %) / 100 % = 30.75 % Al in test item

In five different artificial physiological media, dissolved Al levels ranged from 0.005 and 0.71 % of nominal aluminium contained in the respective loading of 100 mg/L cobalt aluminate blue spinel (IPC-2013 -011) depending on solution parameters and test duration.

Concentration of cobalt in artificial media, calculated nominal cobalt concentration and dissolved amount of cobalt.

media and sample	total Co ± SD in method blanks [µg/L]	total Co ±SD in sample vessels [µg/L]	Co ± SD in sample vessels with blank subtraction [µg/L]	calculated nominal Co concentration in [µg/L]#	dissolved amount Co in artificial media [%] normalized for measured background in method blank
ALF 2h	0.07 ± 0.01	6.78 ± 0.22	6.70 ± 0.22	31469	0.02 ± <0.01
ALF 24h	0.08 ± 0.01	12.9 ± 0.37	12.9 ± 0.37	31469	0.04 ± <0.01
ASW 2h	<LOD	<LOD	<LOD	31123	-
ASW 24h	<LOD	1.56 ± 0.08	1.56 ± 0.08	31123	0.01 ± <0.01
GMB 2h	0.02 ± <0.01	0.08 ± 0.01	0.06 ± 0.01	31396	<0.001
GMB 24h	0.03 ± 0.01	0.40 ± 0.02	0.37 ± 0.02	31396	0.001 ± <0.001
GST 2h	<LOD	4.96 ± 0.16	4.96 ± 0.16	31517	0.02 ± <0.01
GST 24h	<LOD	9.15 ± 0.08	9.15 ± 0.08	31517	0.03 ± <0.01
PBS 2h	<LOD	0.05 ± 0.02	0.05 ± 0.02	31399	<0.001
PBS 24h	<LOD	0.46 ± 0.14	0.46 ± 0.14	31399	0.002 ± <0.001

# (initial weight (e.g. 50 mg) * 30.99 § (percentage cobalt in test item) / 100) * 2 (multiplication to calculate cobalt amount in one litre -> 100 mg/L) = nominal cobalt concentration in [mg/L]/1000 = nominal cobalt concentration in [µg/L]

§according to CoA 39.40 % Co as CoO => 78.65 % cobalt in CoO => (39.40 % * 78.65 %) / 100 % = 30.99 % Co in test item

In five different artificial physiological media, dissolved Co levels ranged from 0.001 to 0.04 % of nominal cobalt contained in the respective loading of 100 mg/L cobalt aluminate blue spinel (IPC-2013-011) depending on solution parameters and test duration.

Mass balance calculation

Total dissolved cobalt concentrations in vessels, filters and syringes measured by ICP-OES indicate an incomplete dissolution of the test item in all physiological media after addition of aqua regia to the sample vessels.

Calculation of cobalt mass balance

media	value for dissolved Co after addition of aqua regia[mg]	nominal concentration [mg] #	recovery [%]
ALF 24h A	0.005	15.7	0.03
ALF 24h B	0.005	15.8	0.03
ASW 24h A	-	-	-
ASW 24h B	0.02	15.6	0.14
GMB 24h A	0.005	15.7	0.03
GMB 24h B	0.005	15.7	0.03
GST 24 A	0.01	15.8	0.04
GST 24h B	0.01	15.8	0.04
PBS 24h A	0.01	15.6	0.03
PBS 24h B	0.01	15.8	0.03

# nominal concentration Co = 39.40 % as CoO in test item = 78.65 % Co in CoO => 15.4937 mg Co in 50 mg test item => 15.4937 * initial weight / 50 mg

Applicant's summary and conclusion

Conclusions:

The bioaccessibility of cobalt aluminate blue spinel 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 dissolved Co and Al concentrations were below 12.9 µg/L and 223 µg/L, respectively, even at the highest loading of 0.1 g/L, referring to a solubility of 0.013 % and 0.223 %, respectively, the pigment is considered biologically inert.

Executive summary:

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

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

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

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

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

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

In total dissolved Co and Al concentrations were below 12.9 µg/L and 223 µg/L, respectively, even at the highest loading of 0.1 g/L, referring to a solubility of 0.013 % and 0.223 %, respectively, the pigment is considered biologically inert.