Olivine, cobalt silicate blue

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:

2015-09-25 to 2015-11-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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: single loading 2 g/L, measurement after filtration, sampling after 2 and 24 hours agitation (100 rpm) at 37 ± 2 °C, three replicates, two method blanks / artificial media (ALF, GMB, GST)

Principles of method if other than guideline:

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.
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.

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2011-02-07

Test material

Test material information

Constituent 1

Reference substance name:

Olivine, cobalt silicate blue

EC Number:

269-093-5

EC Name:

Olivine, cobalt silicate blue

Cas Number:

68187-40-6

Molecular formula:

Co2SiO4

IUPAC Name:

silicon(4+) bis(λ²-cobalt(2+)) tetraoxidandiide

Test material form:

solid: particulate/powder

Details on test material:

- Test item identification: Olivine, cobalt silicate blue (Pigment Blue 73)
- Substance type: inorganic pigment
- Storage condition of test material: Keep dry and container tightly closed. Keep container in an adequately ventilated storage

Specific details on test material used for the study:

Appearance: dark violet powder, odourless
Storage conditions: Store in a dry place.

Test animals

Species:

other: in vitro (simulated human body fluids)

Administration / exposure

Details on exposure:

Test principle in brief:
- three different artificial physiological media,
- single loading of test substance of ~ 2 g/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) of total dissolved cobalt concentrations after filtration
- the study was performed in triplicate

The aim of this test was to assess the dissolution of Olivine, cobalt silicate blue (IPC-2013-006-07) in three artificial physiological media: Artificial lysosomal fluid (ALF, pH= 4.5), Gamble´s solution (GMB, pH= 7.4), Artificial gastric fluid (GST, pH= 1.5). 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 gastro-intestinal tract and by inhalation.

Duration and frequency of treatment / exposure:

Samples were taken after 2h and 24h.

Doses / concentrations

Dose / conc.:

2 other: g of the test item /L artificial physiological 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 element standards were used as cobalt standard (Merck Certipur Cobalt ICP standard 1000 mg/L lot no. HC41722713; Darmstadt, Germany; Merck).
- Certified reference materials: quality control standards TM-25.4 (lot no. 0914), TMDA-53.3 (lot no. 0914) and TMDA-54.5 (lot no. 0815) obtained from Environment Canada and multielement standards (CPI 19 Elements, lot no. 15C267, Netherlands; Merck XXI, lot no. HC55190098, Germany)

Instrumental and analytical set-up for the ICP-OES instrument:
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: 1100W
Wavelengths: Co 230.786 nm and 231.406 nm

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

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: Co 228.616 nm, 230.786 nm and 231.160 nm

The applied LOD/LOQ calculations for the Thermo IRIS Intrepid 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).

Calibration:
- high calibration: blank, 0.5 mg/L, 1.5 mg/L, 2.5 mg/L, 3.5 mg/L, 4.5 mg/L and 5.5 mg/L
- medium calibration: blank, 100 μg/L, 250 μg/L, 500 μg/L, 750 μg/L, 1000 μg/L, 1250 μg/L, 1500 μg/L, 1750 μg/L and 2000 μg/L
- low calibration: blank, 1 μg/L, 2.5 μg/L, 5 μg/L, 7.5 μg/L, 10 μg/L, 25 μg/L 50 μg/L, 75 μg/L, 100 μg/L, 250 μg/L, 500 μg/L, 750 μg/L and 1000 μg/L.

Deviations:
Due to a mistake two vessels of the vessel mass balance for ALF medium were disposed before a sample was taken. According to the results of the vessel mass balance for the media GMB, GST and the remaining ALF medium (one vessel) a total dissolution in aqua regia was measured. After discussion with the study monitor it was decided that the ALF test will not be repeated, because the remaining two vessels are sufficient and this deviation was considered as not critical.

Details on dosing and sampling:

Loading:
The nominal loading in this test was 2 g/L. However, due to weighing uncertainties the actual loadings range from 2.0002 g/L to 2.0016 g/L in the test vessels.

Results and discussion

Main ADME results

Type:

other: Bioaccessibility

Results:

Highest dissolution of Cobalt (loading: 0.1g/L, after 24h) in GST: 874 ± 48.8 mg/L, GMB: 7940 ± 1261 μg/L, ALF: 728 ± 61.0 mg/L

Any other information on results incl. tables

Method validation summary ICP-OES

Validation parameter	Results	Comment
Selectivity	similar data with different wavelengths for ICP-OES method	-
Linearity	applied calibration functions were linear	correlation coefficient at least 0.999648
Limit of detection	Co: 0.48 μg/L (Agilent 720) Co: 0.98 μg/L – 0.07 mg/L (Thermo IRIS Intrepid)
Limit of quantification	Co: 1.45 μg/L (Agilent 720) Co: 3.25 μg/L – 0.23 mg/L (Thermo IRIS Intrepid)
Method blanks Summary	The cobalt concentration in method blanks was below LOD in all samples and artificial media
Accuracy measurement / Reproducibility GMB medium sample measurements	TM-25.4 (dilution factor 2; 13.8 μg Co/L) Co: 111 ± 2.2 % (n = 6)	Low concentration range (undiluted 27.5 μg Co/L – diluted 13.8 μg Co/L)
Accuracy measurement / Reproducibility ALF method blank sample measurements	TM-25.4 (dilution factor 10; 2.75 μg Co/L) Co: 94.0 ± 3.4 % (n = 7)	Low concentration range (undiluted 27.5 μg Co/L – diluted 2.75 μg Co/L)
Accuracy measurement / Reproducibility ALF method blank sample measurements	TMDA-53.3 (dilution factor 10; 25.1 μg Co/L) Co: 101 ± 3.5 % (n = 7)	Mid concentration range (undiluted 251 μg Co/L – diluted 25.1 μg Co/L)
Accuracy measurement / Reproducibility GMB medium sample measurements	TMDA-53.3 (dilution factor 4; 62.8 μg Co/L) Co: 106 ± 3.9 % (n = 6)	Mid concentration range (undiluted 251 μg Co/L – diluted 62.8 μg Co/L)
Accuracy measurement / Reproducibility ALF 2h and 24h (both adapted calibrations), mass balance vessel/filters and GMB mass balance samples (both adapted calibrations)	TMDA-54.5 (318 μg Co/L) Co: 101 ± 1.3 % (n = 28)	Low concentration range
Trueness ALF method blank sample measurements	Quality control standard (Merck XXI 200 μg Co/L) Co: 98.2 ± 4.0 % (n = 7)	High concentration range
Trueness ALF 2h and 24h sample (both adapted calibration), mass balance vessel/filter samples and GMB mass balance samples (both adapted calibrations) measurements	Quality control standard (CPI 500 μg Co/L) Co: 100 ± 1.7 % (n = 28)	Mid concentration range
Trueness GST medium sample and mass balance sample measurements	Quality control standard (CPI 2.5 mg Co/L) Co: 98.2 ± 0.7 % (n = 6)	Mid concentration range
Trueness GMB medium sample measurements	Recalibration standard (50 μg Co/L) Co: 98.7 ± 2.6 % (n = 6)	Mid concentration range
Trueness ALF method blank sample measurements	Recalibration standard (100 μg Co/L) Co: 97.8 ± 2.7 % (n = 7)	Mid concentration range
Trueness GMB mass balance vessel samples, ALF 2h and 24h sample (both adapted calibrations) and GST medium measurements	Recalibration standard (1000 μg Co/L) Co: 102 ± 1.8 % (n = 30)	High concentration range for GMB, ALF and low concentration range for GST samples
Trueness test samples and mass balance	Fortification of samples: Co: 91.1 – 104 %

Solution pH values

The target pH in all media before addition of test substance was in the nominal range. During the study, the pH of GST solutions remained mainly stable in the method blank vessels (1.54 up to 1.56 and 1.58, start of the test to end of the test) but in the test vessels the pH increased from 1.51 up to 2.46, 2.79 and 3.11. Therefore, an effect of the test substance cannot be excluded.

The pH of ALF solutions remained mainly stable. In the method blanks the pH was between 4.50 – 4.52 (start of the test to end of the test), a low increase of the pH in test vessel was measurable (4.50 to 4.58). Therefore, as in the GST medium, a low effect of the test substance cannot be excluded.

In GMB medium, the pH in all vessels (including method blanks) increased during the time of the test from 7.40 to 8.65, 8.68 and 8.71 (test vessels) and 7.40 to 8.69 and 8.76 (method blank vessels). Therefore, an effect of the test substance can be excluded. In fact, the pH of the GMB media does not seem to be stable under the conditions of the test.

Temperature control

The test was performed in an incubated laboratory shaker (Shaking incubation cabinet, Minitron, INFORS AG, Bottmingen, Switzerland) at 100 rpm. The temperature was adjusted to 37.5 in a thermostatically controlled shaking cabinet to reach a temperature of 37 ± 2 °C in the media.

Analytical measurements

The nominal loading in this test was 1000 mg/ 500mL. However, due to weighing uncertainties the actual loadings range from 2.0002 g/L to 2.0016 g/L in the 9 test vessels.

Concentration of dissolved cobalt in artificial physiological media.

Media and sample	LOD/LOQ of Co measurement series [μg/L]; [mg/L]	total Co ± SD in method blanks [μg/L]; [mg/L]	total Co ± SD in sample vessels [μg/L]; [mg/L]	total Co ± SD in sample vessels [μg/L]; [mg/L] (corrected concentrations, corresponding to a loading of exactly 2 g/L)
GST 2h	LOD: 0.07 mg/L LOQ: 0.23 mg/L	all method blanks below LOD	192 ± 89.4 mg/L	192 ± 89.3 mg/L
GST 24h	LOD: 0.07 mg/L LOQ: 0.23 mg/L	all method blanks below LOD	874 ± 48.8 mg/L	874 ± 48.8 mg/L
GMB 2h	LOD: 0.98 μg/L LOQ: 3.25 μg/L	all method blanks below LOD	7170 ± 6755 μg/L	7169 ± 6754 μg/L
GMB 24h	LOD: 0.98 μg/L LOQ: 3.25 μg/L	all method blanks below LOD	7943 ± 1260 μg/L	7940 ± 1261 μg/L
ALF 2h	ALF method blanks :# LOD: 0.48 μg/L LOQ: 1.45 μg/L ALF 2h test samples:$ LOD: 13.3 μg/L LOQ: 44.5 μg/L (only sample vessel 1 2h) LOD: 27.9 μg/L LOQ: 92.8 μg/L	all method blanks below LOD	113 ± 43.5 mg/L	113 ± 43.5 mg/L
ALF 24h	ALF method blanks :# LOD: 0.48 μg/L LOQ: 1.45 μg/L ALF 24h test samples:$ LOD: 27.9 μg/L LOQ: 92.8 μg/L	all method blanks below LOD	728 ± 60.9 mg/L	728 ± 61.0 mg/L

# measurement performed with ICP-OES Agilent 720

$ measurement performed with ICP-OES IRIS Intrepid (two LOD/LOQ for 2h samples because of adapted calibration)

Mass balance calculation

The mass balance dissolved cobalt concentrations in vessels and filters as measured by ICP-OES indicate no complete dissolution of IPC-2013-006-07 in the physiological media after addition of aqua regia to the sample vessels. Due to the observations during the mass balance a small amount of particles were still visible which could be a result of insoluble parts of the test item (for example according to CoA 34.2% SiO2 in test item

Amount of cobalt in mass balance samples (vessels, filter/syringes and taken samples) for each media.

media	value for dissolved Co after addition of aqua regia		In samples [mg]	Calculated value Co [mg]*	Nominal amount [mg] #	Recovery [%]
	Vessels [mg]	Filters / syringes [mg]
GST vessel 1	491	0.41	36.4	528	507	104
GST vessel 2	485	0.40	46.4	532	507	105
GST vessel 3	478	0.42	45.1	523	507	103
GMB vessel 1	533	0.36	0.49	533	507	105
GMB vessel 2	525	0.18	0.35	526	507	104
GMB vessel 3	538	1.00	0.97	540	507	106
ALF vessel 1	470	0.61	60.6	503	507	99.1
ALF vessel 2	Due to a mistake vessel 2 and 3 of mass balance were disposed before sample was taken see amendment 2
ALF vessel 3

* Measured dissolved Co includes the amount of cobalt in test solutions after addition of aqua regia, the amount of cobalt rinsed of used filters and syringe and the amount of cobalt removed with samples during the study.

Example: 491 mg (amount Co in vessels) + 0.41 mg (amount Co in filters/syringes) + 36.4 mg (amount Co in samples) = 528 mg

# nominal amount Co = 50.7 % in test item (according to CoA)-> 507 mg Co in 1 g test item -> nominal amount 507 mg * initial weight / 1 g

Example: 507 mg Co * 1000.3 mg test item / 1000 mg = 507 mg Co

Applicant's summary and conclusion

Conclusions:

The bioaccessibility of Cobalt silcate olivine has been investigated experimentally in vitro by simulating dissolution under physiological conditions considered to mimic the most relevant exposure routes (oral and inhalation). At a loading of 2 g test item /L dissolved Co concentrations after 24 h in GST, GMB and ALF were 874 mg/L (192 mg/L after 2 h), 7940 µg/L and 728 mg/L, respectively, referring to a solubility of ~ 43.7 % (9.6 % after 2h), 0.4 % and ~ 36.4 %, respectively.
The dissolved Co concentrations (according to the CoRC bioaccessibility protocol) were 192 mg/L at a 2 g loading, corresponding to a solubility of 9.6 % in the most effective surrogate physiological medium (GST, 2 hours).

Total Co ± SD in sample vessels (corrected concentrations, corresponding to a loading of exactly 2 g/L)
- GST 2h: 192 ± 89.3 mg/L
- GST 24h: 874 ± 48.8 mg/L
- GMB 2h: 7169 ± 6754 μg/L
- GMB 24h: 7940 ± 1261 μg/L
- ALF 2h: 113 ± 43.5 mg/L
- ALF 24h:728 ± 61.0 mg/L