Reaction mass of cobalt olivine and crystalline silicon dioxide

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 1

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Reference 2

Reference Type:

other: Amendment

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:

Reaction mass of cobalt olivine and crystalline silicon dioxide

EC Number:

701-439-7

Molecular formula:

Co2SiO4∙xSiO2 (0,7≤x≤1,5)

IUPAC Name:

Reaction mass of cobalt olivine and crystalline silicon dioxide

Test material form:

solid: particulate/powder

Details on test material:

- Test item identification: Olivine, cobalt silicate blue (Pigment blue 73)
- new chemical name: Reaction mass of cobalt olivine and crystalline silicon dioxide
- Substance type: inorganic pigment
- Storage conditions: Keep dry and container tightly closed. Keep container in an adequately ventilated storage

Specific details on test material used for the study:

Appearance: light 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-2013-006-01) 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) were analysed for total dissolved cobalt by ICP-OES along with the samples to determine the accuracy of the applied analytical method. To meet quality assurance requirements recovery needs to be in the range of ± 15 % of the respective certified value. Furthermore the calibration solutions were measured along with the ICP-OES measurements as recalibration standards.

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: 1200W
- 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, 5.5 mg/L, 6.5 mg/L and 7.5 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 2.0000 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: 808 ± 32.3 mg/L, GMB: 197 ± 17.2 μg/L, ALF: 529 ± 20.2 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	In all samples below LOD	-
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 sample measurements and GMB mass balance samples (both adapted calibrations)	TMDA-54.5 (318 µg Co/L) Co: 101 ± 1.2 % (n = 18)	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 sample and GMB mass balance samples (both adapted calibrations) measurements	Quality control standard (CPI 500 µg Co/L) Co: 101 ± 1.8 % (n = 18)	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)	High 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 measurements	Recalibration standard (100 µg Co/L) Co: 97.8 ± 2.7 % (n = 7)	Mid concentration range
Trueness GST medium sample and mass balance sample measurements; mass balance vessel samples GMB and ALF samples and mass balance measurements	Recalibration standard (1000 μg Co/L) Co: 102 ± 2.1 % (n = 20)	Low concentration range for GST medium, High concentration range for GMB and ALF samples
Trueness test samples and mass balance	Fortification of samples: Co: 95.9 – 104 %

Solution pH values

After preparation of the artificial physiological media, solution pH was adjusted to their respective target pH. 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 but in the test vessels the pH increased from 1.51 – 1.52 up to 2.31 – 2.52. 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 4.59). 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 8.88 (test vessels) and 7.4 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 temperature was adjusted to 37.5 in a thermostatically controlled shaking cabinet to reach a temperature of 37 ± 2 °C in the media. The temperature in thermostatically controlled shaking cabinet was confirmed by a certified thermometer and a thermo data logger. In all test vessels, the temperature was constant at 37 ± 2 °C.

Analytical measurements

The nominal loading in this test was 1000 mg/ 500mL. However, due to weighing uncertainties the actual loadings range from 2.0000 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	88.1 ± 39.3 mg/L	88.0 ± 39.3 mg/L
GST 24h	LOD: 0.07 mg/L LOQ: 0.23 mg/L	all method blanks below LOD	808 ± 32.5 mg/L	808 ± 32.3 mg/L
GMB 2h	LOD: 0.98 μg/L LOQ: 3.25 μg/L	all method blanks below LOD	110 ± 37.4 μg/L	110 ± 37.4 μg/L
GMB 24h	LOD: 0.98 μg/L LOQ: 3.25 μg/L	all method blanks below LOD	197 ± 17.2 μg/L	197 ± 17.2 μg/L
ALF 2h	ALF method blanks: LOD: 0.48 μg/L LOQ: 1.45 μg/L ALF test samples LOD: 27.9 μg/L LOQ: 92.8 μg/L	all method blanks below LOD	106 ± 16.5 mg/L	106 ± 16.4 mg/L
ALF 24h	ALF method blanks: LOD: 0.48 μg/L LOQ: 1.45 μg/L ALF test samples LOD: 27.9 μg/L LOQ: 92.8 μg/L	all method blanks below LOD	530 ± 20.3 mg/L	529 ± 20.2 mg/L

Mass balance calculation

The mass balance dissolved cobalt concentrations in vessels and filters as measured by ICP-OES indicate a complete dissolution of IPC-2013-006-01 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 45.2 % 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/L]	Filters/ syringes [mg]
GST vessel 1	394	0.17	38.4	433	415	104
GST vessel 2	391	0.12	36.4	428	415	103
GST vessel 3	349	0.13	32.8	381	414	92.0
GMB vessel 1	427	0.18	0.01	427	415	103
GMB vessel 2	433	0.28	0.01	433	415	104
GMB vessel 3	442	0.28	0.01	442	415	107
ALF vessel 1	369	1.70	23.9	395	415	95.2
ALF vessel 2	Due to a mistake vessel of mass balance was disposed before sample was taken
ALF vessel 3	369	1.98	26.8	398	415	95.9

* 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: 394 mg (amount Co in vessels) + 0.17 mg (amount Co in filters/syringes) + 38.4 mg (amount Co in samples) = 433 mg

# nominal amount CoO = 52.70 % in test item (according to CoA) -> 78.6483 % Co in CoO -> 414 mg Co in 1 g test item -> nominal amount 414 mg * initial weight / 1 g

Example: 414 mg Co * 1000.3 mg test item / 1000 mg = 415 mg Co

Applicant's summary and conclusion

Conclusions:

The bioaccessibility of Cobalt silicate olivine and silca polymorphs 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 808 mg/L (88 mg/L after 2 h), 0.197 mg/L and 529 mg/L, respectively, referring to a solubility of ~ 40.4 % (4.41 % after 2h), 0.01 % and ~ 26.45 %, respectively.
The dissolved Co concentrations (according to the CoRC bioaccessibility protocol) were 88 mg/L at a 2 g loading, corresponding to a solubility of 4.41 % 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: 88.0 ± 39.3 mg/L
- GST 24h: 808 ± 32.3 mg/L
- GMB 2h: 110 ± 37.4 μg/L
- GMB 24h: 197 ± 17.2 μg/L
- ALF 2h: 106 ± 16.4 mg/L
- ALF 24h: 529 ± 20.2 mg/L