Reaction mass of Willemite, white and zinc iron chromite brown spinel

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics in vitro / ex vivo

Remarks:

transformation/dissolution in artificial physiological media / bioaccessibility

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-09-24 to 2013-02-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Objective of study:

bioaccessibility (or bioavailability)

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 2 h and 24 h; determination of Al, Cr, Fe and Zn concentrations after filtration by ICP-OES.

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

Species:

other: in vitro (simulated human body fluids)

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) of dissolved aluminium, chromium, iron and zinc 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-2013-017 (Reaction mass of willemite, white and zinc iron chromite brown 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.

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 were used as aluminium, iron, chromium and zinc standards (Merck XXl lot no. HC075495, Merck Certipur Aluminium ICP standard 1000 mg/L lot no. HC125228; Merck Certipur Chromium ICP standard 1000 mg/L lot no. HC114885, Merck Certipur Iron ICP standard 1000 mg/L lot no. HC093237, Merck Certipur Zinc ICP standard 1000 mg/L lot no. HC090981; Darmstadt, Germany).
- Certified reference materials: TMDA-70 (lot no. 0310) and TM-15.2 (lot no. 1011) obtained from Environment Canada and a multielement standard (Merck VIII, lot no. HC137073, 19 elements, CPI, Amsterdam, the Netherlands, lot.no. 12A089)

Instrumental and analytical set-up for the ICP-OES instrument (sample 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: Al: 167.019 nm, 236.705 nm, 308.215 nm and 396.152 nm; Cr: 205.560 nm, 206.158 nm, 266.602 nm, 284.325 nm and 357.868 nm; Fe: 234.350 nm, 238.204 nm, 238.863 nm and 258.588 nm; Zn: 202.548 nm, 206.200 nm and 213.857 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

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: Fe: 238.204 nm and 239.562 nm; Zn: 202.548 nm, 206.200 nm and 213.856 nm
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).

- Calibration: blank, 1, 5, 7.5, 10, 25, 50, 75, 100, 200, 250, 300, 400 and 500 µg/L. Calibrations were performed before each measurement.
- Correlation factors (r): at least 0.9988

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 100.628 mg/L to 101.96 mg/L in the test vessels.

Type:

other: Bioaccessibility

Results:

Higest dissolution at a loading of 0.1g/L in ALF after 24h: Al = 20.7± 0.21 µg/L, Cr = 2.74 ± 0.11 µg/L, Fe = 69.8 ± 1.60 µg/L, Zn = 26761 ± 789 µg/L

Type:

other: Bioaccessibility

Results:

Higest dissolution at a loading of 0.1g/L in GST after 24h: Al = 32.4 ± 0.72 µg/L, Cr = 3.21 ± 0.05 µg/L, Fe = 92.7 ± 0.54 µg/L, Zn = 26411 ± 1718 µg/L

Method validation summary (ICP-OES)

validation parameter	results	Comment
Selectivity	similar data with two different wavelengths for ICP-OES method	No interference observed
Linearity	applied calibration functions were linear	correlation coefficient at least 0.9988
Limit of detection	Al: 0.15 – 0.31 µg/L Cr: 0.18 – 0.32 µg/L Fe: 0.33 – 9.48 µg/L Zn: 0.03 – 8.48 µg/L
Limit of quantification	Al: 0.46 – 0.92 µg/L Cr: 0.54 – 0.95 µg/L Fe: 1.02 – 31.6 µg/L Zn: 0.09 – 28.3 µg/L
Method blanks	see table: `Concentration of method blanks in artificial media´	Elevated concentrations of Al and Fe in method blanks origin from the applied chemicals for preparation of different media
Accuracy samples	mean recovery for CRM TMDA-70: Al: 103 ± 2.8 % (n = 14) Cr: 102 ± 0.8 % (n = 14) Fe: 100 ± 0.8 % (n = 14) Zn: 103 ± 1.2 % (n = 17)	high concentration range (415 µg Al/L; 389 µg Cr/L, 368 µg Fe/L, 477 µg Zn/L)
Accuracy samples	mean recovery for CRM TM15.2: Al: 105 ± 2.4 % (n = 14) Cr: 107 ± 1.1 % (n = 14) Fe: 103 ± 1.4 % (n = 14) Zn: 111 ± 1.2 % (n = 17)	low concentration range (33.9 µg Al/L; 16.4 µg Cr/L, 25.8 µg Fe/L, 35.4 µg Zn/L)
Accuracy mass balance	mean recovery for CRM TMDA-70: Fe: 104 ± 10.1 % (n = 9) Zn: 98.5 ± 4.8 % (n = 7)	high concentration range (368 µg Fe/L, 477 µg Zn/L)
Trueness measurement series in samples	mean recovery for recalibration standard : Al: 104 ± 1.6 % (n = 11) Cr: 97.6 ± 1.0 % (n = 11) Fe: 98.8 ± 0.9 % (n = 11) Zn: 105 ± 1.1 % (n = 11)	mid concentration range (50 µg/L)
Trueness measurement series in samples	mean recovery for recalibration standard : Al: 98.1 ± 1.1 % (n = 3) Cr: 96.8 ± 0.8 % (n = 3) Fe: 100 ± 0.8 % (n = 3) Zn: 101 ± 2.1 % (n = 6)	mid high concentration range (100 µg/L)
Trueness mass balance samples	mean recovery for recalibration standard : Fe: 100 ± 19 % (n = 5)	low concentration range (10 µg/L)
Trueness mass balance samples	mean recovery for recalibration standard : Fe: 95.8 ± 5.5 % (n = 5)	Mid low concentration range (25 µg/L)
Trueness mass balance samples	mean recovery for recalibration standard : Zn: 96.9 ± 0.9 % (n = 5)	low concentration range (50 µg/L)
Trueness mass balance samples	mean recovery for recalibration standard : Fe: 102 ± 2.2 % (n = 4) Zn: 102 ± 3.1 % (n = 7)	Mid concentration range (100 µg/L)
Trueness mass balance samples	mean recovery for recalibration standard : Fe: 102 ± 1.5 % (n = 4) Zn: 98.0 ± 0.7 % (n = 2)	High concentration range (250 µg/L)
Trueness	Fortification of samples: Al: 90.0 – 97.3 % Cr: 88.0 – 99.0 % Fe: 90.3 – 98.4 % Zn: 92.3 – 100 %
Reproducibility sample measurement	mean recovery for CRM TMDA-70: Al: 103 ± 2.8 % (n = 14) Cr: 102 ± 0.8 % (n = 14) Fe: 100 ± 0.8 % (n = 14) Zn: 103 ± 1.2 % (n = 17)	high concentration range (415 µg Al/L; 389 µg Cr/L, 368 µg Fe/L, 477 µg Zn/L)
Reproducibility sample measurement	mean recovery for CRM TM15.2: Al: 105 ± 2.4 % (n = 14) Cr: 107 ± 1.1 % (n = 14) Fe: 103 ± 1.4 % (n = 14) Zn: 111 ± 1.2 % (n = 17)	low concentration range (33.9 µg Al/L; 16.4 µg Cr/L, 25.8 µg Fe/L, 35.4 µg Zn/L)
Reproducibility mass balance	mean recovery for CRM TMDA-70: Fe: 104 ± 10.1 % (n = 9) Zn: 98.5 ± 4.8 % (n = 7)	high concentration range (368 µg Fe/L, 477 µg Zn/L)

Concentration of method blanks in artificial media

Artificial media	Element
	Al	Cr	Fe	Zn
GST	Method blanks above LOQ but at least 2.81 fold lower than concentrations in samples	<LOD	three method blanks 2h below LOQ one above LOQ and 24h blanks above LOQ but at least 25 fold than concentration in samples	Method blanks above LOQ but at least 11403 fold lower than concentrations in samples
GMB	Method blanks above LOQ and mainly the same as the concentration in samples	One method blank 2h below LOQ three above LOQ and mainly the same concentration as in samples; method blanks 24h below LOQ	<LOD/LOQ	<LOD/LOQ
ALF	Method blanks above LOQ but at least 6.14 fold lower than concentrations in samples	Method blanks above LOQ but 1.46 fold lower than concentrations in samples	Method blanks above LOQ but at least 3.10 fold lower than concentrations in samples	three method blanks 2h below LOQ one above LOQ; one method blank 24h below LOD and three above LOQ but at least 38939 fold lower than concentration in samples
ASW	Method blanks above LOQ but 1.49 fold lower than concentrations in samples	<LOD	<LOQ	Method blanks above LOQ but at least 1246 fold lower than concentrations in samples
PBS	Method blanks above LOQ but 1.29 fold lower than concentrations in samples	Mainly < LOQ except one method blank 2h, nearly the same concentration as in samples	Method blanks above LOQ and mainly the same as the concentration in samples	Method blanks above LOQ but at least 98.3 fold lower than concentrations in samples

Measured pH in method blank and test vessels in ALF medium

sample name	target pH	pH prior to the test	temp. [°C] prior to the test	pH after 2h	temp.[°C]	pH after 24h	temp.[°C]
ALF vessel A	4.5	4.5	31.5	4.5	30.7	4.5	28.4
ALF vessel B	4.5	4.5	31.6	4.5	30.8	4.5	28.5
ALF blank vessel A	4.5	4.5	31.0	4.5	33.3	4.5	31.7
ALF blank vessel B	4.5	4.5	31.4	4.5	33.5	4.5	32.0

Measured pH in method blank and test vessels in ASW medium

sample name	target pH	pH prior to the test	temp. [°C] prior to the test	pH after 2h	temp.[°C]	pH after 24h	temp.[°C]
ASW vessel A	6.5	6.5	33.5	7.3	32.2	6.2	30.2
ASW vessel B	6.5	6.5	33.2	7.3	32.4	6.2	30.2
ASW blank vessel A	6.5	6.5	33.3	6.4	34.3	6.0	33.2
ASW blank vessel B	6.5	6.5	33.5	6.4	34.5	5.9	33.6

 

Measured pH in method blank and test vessels in GMB medium

sample name	target pH	pH prior to the test	temp. [°C] prior to the test	pH after 2h	temp.[°C]	pH after 24h	temp.[°C]
GMB vessel A	7.4	7.4	32.6	7.8	31.7	8.8	29.8
GMB vessel B	7.4	7.4	32.5	7.7	31.6	8.8	29.7
GMB blank vessel A	7.4	7.4	31.8	7.7	33.9	8.8	32.7
GMB blank vessel B	7.4	7.4	32.5	7.6	34.1	8.8	33.1

 

Measured pH in method blank and test vessels in GST medium

sample name	target pH	pH prior to the test	temp. [°C] prior to the test	pH after 2h	temp.[°C]	pH after 24h	temp.[°C]
GST vessel A	1.5 – 1.6	1.6	34.4	1.6	32.5	1.6	31.6
GST vessel B	1.5 – 1.6	1.6	34.4	1.6	32.5	1.6	31.6
GST blank vessel A	1.5 – 1.6	1.6	34.4	1.6	35.1	1.6	34.5
GST blank vessel B	1.5 – 1.6	1.6	34.5	1.6	34.9	1.6	33.8

 

Measured pH in method blank and test vessels inPBS medium

sample name	target pH	pH prior to the test	temp. [°C] prior to the test	pH after 2h	temp.[°C]	pH after 24h	temp.[°C]
PBS vessel A	7.2 - 7.4	7.4	34.4	7.4	32.9	7.4	30.7
PBS vessel B	7.2 - 7.4	7.4	34.2	7.4	32.5	7.4	30.2
PBS blank vessel A	7.2 - 7.4	7.4	34.0	7.4	34.8	7.4	33.8
PBS blank vessel B	7.2 - 7.4	7.4	34.4	7.4	34.9	7.4	34.1

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	2.40 ± 0.10	14.7 ± 2.32	12.3 ± 2.32	5760	0.21 ± 0.04
ALF 24h	2.93 ± 0.01	23.6 ± 0.21	20.7 ± 0.21	5760	0.36 ± <0.01
ASW 2h	2.58 ± 0.04	4.59 ± 0.28	2.01 ± 0.28	5729	0.04 ± 0.01
ASW 24h	3.23 ± 0.15	4.82 ± 0.34	1.59 ± 0.34	5729	0.03 ± 0.01
GMB 2h	5.09 ± 0.12	6.53 ± 0.74	1.44 ± 0.74	5727	0.03 ± 0.01
GMB 24h	5.46 ± 0.04	5.59 ± 0.06	0.42 ± 0.48	5727	0.01 ± 0.01
GST 2h	7.24 ± 0.47	20.3 ± 7.34	13.1 ± 7.34	5734	0.23 ± 0.13
GST 24h	8.94 ± 0.60	41.4 ± 0.72	32.4 ± 0.72	5734	0.57 ± 0.01
PBS 2h	3.02 ± 0.10	3.89 ± 0.12	0.87 ± 0.12	5723	0.02 ± <0.01
PBS 24h	3.85 ± 0.42	4.53 ± 0.78	0.80 ± 0.60	5723	0.01 ± 0.01

# (initial weight (e.g. 50 mg)* 5.66§ (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 10.70 % Al as Al₂O₃ -> 52.93 % aluminium in Al₂O₃ -> (10.70 % * 52.93 %) / 100 % = 5.66 % Al in test item

In five different artificial physiological media, between 0.01 and 0.57 % of aluminium was dissolved from the test item IPC-2013-017 (reaction mass of willemite, white and zinc iron chromite brown spinel) depending on solution parameters and test duration.

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

media and sample	total Cr ± SD in method blanks [µg/L]	total Cr ±SD in sample vessels [µg/L]	Cr ± SD in sample vessels with blank subtraction [µg/L]	calculated nominal Cr concentration in [µg/L]#	dissolved amount Cr in artificial media [%] normalized for measured background in method blank
ALF 2h	3.80 ± 0.03	5.54 ± 0.12	1.75 ± 0.12	6269	0.03 ± <0.01
ALF 24h	3.93 ± 0.02	6.67 ± 0.11	2.74 ± 0.11	6269	0.04 ± <0.01
ASW 2h	<LOD	0.57	0.57	6235	0.01
ASW 24h	<LOD	1.03 ± 0.08	1.03 ± 0.08	6235	0.02 ± <0.01
GMB 2h	0.63 ± 0.02	0.60 ± 0.02	-	6233	-
GMB 24h	<LOQ	0.73 ± 0.08	0.73 ± 0.08	6233	0.01 ± <0.01
GST 2h	<LOD	1.05 ± 0.08	1.05 ± 0.08	6241	0.02
GST 24h	<LOD	3.21 ± 0.05	3.21 ± 0.05	6241	0.05 ± <0.01
PBS 2h	0.56	0.63 ± 0.05	0.15 ± 0.07	6228	0.002 ± 0.001
PBS 24h	<LOQ	0.66 ± 0.07	0.66 ± 0.07	6228	0.01 ± <0.01

# (initial weight (e.g. 50 mg) * 6.16 § (percentage chromium in test item) / 100) * 2 (multiplication to calculate chromium amount in one litre -> 100 mg/L) = nominal chromium concentration in [mg/L]/1000 = nominal chromium concentration in [µg/L]

§ according to CoA 9.01 % Cr as Cr₂O₃ -> 68.42 % chromium in Cr₂O₃ -> (9.01 % * 68.42 %) / 100 % = 6.16 % Cr in test item

In five different artificial physiological media, between 0.002 and 0.05 % of chromium was dissolved from the test item IPC-2013-017 (reaction mass of willemite, white and zinc iron chromite brown spinel) depending on solution parameters and test duration.

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

media and sample	total Fe ± SD in method blanks [µg/L]	total Fe ±SD in sample vessels [µg/L]	Fe ± SD in sample vessels with blank subtraction [µg/L]	calculated nominal Fe concentration in [µg/L]#	dissolved amount Fe in artificial media [%] normalized for measured background in method blank
ALF 2h	19.3 ± 0.15	59.7 ± 6.95	40.4 ± 6.95	9465	0.43 ± 0.07
ALF 24h	19.9 ± 0.06	89.7 ± 1.60	69.8 ± 1.60	9465	0.74 ± 0.02
ASW 2h	<LOQ	2.92 ± 0.74	2.92 ± 0.74	9414	0.03 ± 0.01
ASW 24h	<LOQ	4.40 ± 1.15	4.40 ± 1.15	9414	0.05 ± 0.01
GMB 2h	<LOD/LOQ	2.24	2.42	9411	0.02
GMB 24h	<LOD/LOQ	<LOD/LOQ	<LOD/LOQ	9411
GST 2h	1.42	34.8 ± 18.7	33.4 ± 18.7	9422	0.35 ± 0.20
GST 24h	1.24 ± 0.03	93.9 ± 0.54	92.7 ± 0.54	9422	0.98 ± 0.01
PBS 2h	3.91 ± 0.02	6.71 ± 2.95	2.80 ± 2.95	9403	0.03 ± 0.03
PBS 24h	4.71 ± 0.66	4.70 ± 0.10	0.37	9403	0.003

# (initial weight (e.g. 50 mg)* 9.30 § (percentage iron in test item)/ 100) *2 (multiplication to calculate iron amount in one litre -> 100 mg/L) = nominal iron concentration in [mg/L]/1000 = nominal iron concentration in [µg/L]

§ according to CoA 13.30 % Fe as Fe₂O₃ -> 69.94 % iron in Fe₂O₃ -> (13.30 % * 69.94 %) /100 % = 9.30 % Fe in test item

In five different artificial physiological media, between 0.003 and 0.98 % of iron was dissolved from the test item IPC-2013-017 (reaction mass of willemite, white and zinc iron chromite brown spinel) depending on solution parameters and test duration.

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

media and sample	total Zn ± SD in method blanks [µg/L]	total Zn ±SD in sample vessels [µg/L]	Zn ± SD in sample vessels with blank subtraction [µg/L]	calculated nominal Zn concentration in [µg/L]#	dissolved amount Zn in artificial media [%] normalized for measured background in method blank
ALF 2h	0.39	15014 ± 3480	15014 ± 3480	43497	34.5 ± 8.00
ALF 24h	0.40 ± 0.01	26761 ± 789	26761 ± 789	43497	61.5 ± 1.81
ASW 2h	0.71 ± 0.01	878 ± 110	878 ± 110	43263	2.03 ± 0.25
ASW 24h	0.77 ± 0.22	1678 ± 181	1677 ± 181	43263	3.88 ± 0.42
GMB 2h	<LOD/LOQ	90.7 ± 3.05	90.7 ± 3.05	43249	0.21 ± 0.01
GMB 24h	<LOD/LOQ	2.25	2.25	43249	0.01
GST 2h	0.99 ± 0.44	12741 ± 6747	12740 ± 6747	43301	29.4 ± 15.6
GST 24h	2.31 ± 0.02	26413 ± 1718	26411 ± 1718	43301	61.0 ± 3.97
PBS 2h	1.60 ± 0.10	158 ± 38.5	156 ± 38.5	43214	0.36 ± 0.09
PBS 24h	1.62 ± 0.11	313 ± 2.77	311 ± 2.77	43214	0.72 ± 0.01

# (initial weight (e.g. 50 mg) * 42.74§ (percentage zinc in test item) / 100) * 2 (multiplication to calculate zinc amount in one litre -> 100 mg/L) = nominal zinc concentration in [mg/L] / 1000 = nominal zinc concentration in [µg/L]

§ according to CoA 53.20 % Zn as ZnO -> 80.34 % zinc in ZnO -> (53.20 % * 80.34 %) / 100 % = 42.74 % Zn in test item

In five different artificial physiological media, between 0.21 and 61.5 % of zinc was dissolved from the test item IPC-2013-017 (reaction mass of willemite, white and zinc zinc chromite brown spinel) depending on solution parameters and test duration.

Mass balance calculation

Total dissolved iron and zinc 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 iron mass balance

media	value for dissolved Fe after addition of aqua regia [mg]	nominal concentration [mg] #	recovery [%]
ALF 24h A	0.05	4.74	1.15
ALF 24h B	0.06	4.72	1.20
ASW 24h A	0.05	4.68	1.07
ASW 24h B	0.05	4.74	1.09
GMB 24h A	0.05	4.72	1.16
GMB 24h B	0.05	4.69	1.11
GST 24 A	0.05	4.73	1.09
GST 24h B	0.05	4.73	1.08
PBS 24h A	0.06	4.69	1.22
PBS 24h B	0.06	4.71	1.20

# nominal concentration Fe = 13.30 % as Fe₂O₃in test item = 36.94 % Fe in Fe₂O₃ => 4.651 mg Fe in 50 mg test item => 4.651 * initial weight / 50 mg

Calculation of zinc mass balance

media	value for dissolved Zn after addition of aqua regia [mg]	nominal concentration [mg] #	recovery [%]
ALF 24h A	11.3	21.8	51.7
ALF 24h B	11.0	21.7	50.7
ASW 24h A	12.6	21.5	58.6
ASW 24h B	12.6	21.8	57.9
GMB 24h A	13.4	21.7	61.9
GMB 24h B	12.9	21.5	60.1
GST 24 A	11.2	21.7	51.7
GST 24h B	11.3	21.6	52.5
PBS 24h A	13.4	21.6	61.9
PBS 24h B	13.4	21.6	62.1

# nominal concentration Zn = 53.20 % as ZnO in test item = 80.34 % Zn in ZnO => 21.370 mg Zn in 50 mg test item => 21.370 * initial weight / 50 mg 

Conclusions:

Dissolved Cr, Al, Zn and Fe concentrations were below 3.21 µg Cr/L, 32.4 µg Al/L, 26.7 mg Zn/L and 92.7 µg Fe/L respectively, even at the highest loading of 0.1 g/L, referring to a solubility of 0.003 %, 0.032 %, 26.7 % and 0.093 % respectively.

Executive summary:

The bioaccessibility of reaction mass of willemite, white and zinc iron chromite brown 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 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.

Under the conditions of this test (flasks with different artificial physiological media; loadings of 100 mg/L, 37 °C, sampling after 2h and 24h), the concentrations of dissolved aluminium, chromium, iron and zinc were as follows:

In five different artificial physiological media dissolved aluminium concentrations were between 0.01 and 0.57 %, dissolved chromium concentrations were between 0.002 and 0.05 %, dissolved iron concentrations were between 0.003 and 0.98 %, and dissolved zinc concentrations were between 0.21 and 61.5 % based on respective element contained in 100 mg pigment.

Description of key information

Dissolved Cr, Al, Zn and Fe concentrations were below 3.21 µg Cr/L, 32.4 µg Al/L, 26.7 mg Zn/L and 92.7 µg Fe/L respectively, even at the highest loading of 0.1 g/L, referring to a solubility of 0.003 %, 0.032 %, 26.7 % and 0.093 %, respectively.

Additional information

Discussion on bioaccumulation potential result:

The chemical and physiological properties of the pigment reaction mass of willemite, white and zinc iron chromite brown spinelare 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 1.5), which mimics the very harsh digestion milieu of high acidity in the stomach.

An oral, dermal or inhalative uptake of the substance for humans exposed to the pigment is negligible.

The dissolution of Cr of the test item is between below 0.15 µg/L /0.66 µg/L (PBS) and 1.05 µg/L/ 3.21 µg/L (GST) at a loading of 0.1g/L after 2 and 24 hours.

The dissolution of Al of the test item is in a range of 0.87 µg/L / 0.80 µg/L (PBS) and 13.1 µg/L 32.4 µg/L (GST) at a loading of 0.1 g/L after 2 and 24 hours.

The dissolution of Zn of the test item is in a range of 15.01 mg/L / 26.8 mg/L (GMB) and / 61.8 µg/L (ALF) at a loading of 0.1 g/L after 2 and 24 hours.

The dissolution of Fe of the test item is in a range of 2.42 µg/L / below LoD/LoQ (GMB) and 33.4 µg/L/ 92.7 µg/L (ALF) at a loading of 0.1 g/L after 2 and 24 hours.

A pH dependent dissolution can be observed.

In conclusion, dissolved Cr, Al, Zn and Fe concentrations were below 3.21 µg Cr/L, 32.4 µg Al/L, 26.7 mg Zn/L and 92.7 µg Fe/L respectively, even at the highest loading of 0.1 g/L, referring to a solubility of 0.003 %, 0.032 %, 26.7 % and 0.093 %, respectively.