Spinels, chromium iron manganese brown

Basic toxicokinetics

Administrative data

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:

2012-09-13 to 2012-11-14

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 Cr, Fe and Mn 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:

Spinels, chromium iron manganese brown

EC Number:

271-411-2

EC Name:

Spinels, chromium iron manganese brown

Cas Number:

68555-06-6

Molecular formula:

(Mn, Fe)(Mn, Fe, Cr)2O4

IUPAC Name:

oxo[(oxochromio)oxy]chromium; oxo[(oxoferrio)oxy]iron; oxo[(oxomanganio)oxy]manganese

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material: Spinels, chromium iron manganese brown
- C.I. name: Pigment Brown 46
- Substance type: inorganic pigment
- Physical state:solid, black powder, odourless
- Storage condition of test material: at room temperature, 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 chromium, iron and manganese 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-2013-013 (Spinels, chromium iron manganese brown) 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: multielement standards were used as chromium, iron and manganese standards (Merck XXI lot no. HC075495, Darmstadt, Germany; Merck VIII lot no. HC137073; Merck IV lot no. HC957274; CPI, Amsterdam, The Netherlands, lot no. 12A089).
- Certified reference materials: TMDA-70 (lot no. 0310) and TM15.2 (lot no. 1011 from Environment Canada) and multielement standard CPI ( Amsterdam, The Netherlands, lot no. 12A089), Merck IV (lot no. HC957274) and Merck VIII (lot no. HC137073) Darmstadt, Germany.

The analysis of total dissolved iron in samples and manganese in mass balance samples were measured using an IRIS Intrepid II ICP-OES (Thermo Electron, Dreieich, Germany).
Instrumental and analytical set-up for the ICP-OES instrument:
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, 239.526 nm and 259.940 nm; Mn: 257.610 nm, 259.373 nm and 260.569
Calibration: blank, 1, 1.5, 3.5, 5, 7.5, 10, 25, 50, 75, 100, 200, 250, 300, 400 and 500 µg/L
Correlation coefficients (r) were at least 0.9997
Four measurements were performed for the determination of iron and manganese 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.

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: 52Cr, 54Cr, 55Mn, 103Rh (internal standard)
Calibrations: blank, 0.1, 0.25, 0.5, 0.75, 1.0, 2.5, 5.0, 7.5, 10.0, 25.0L, 50.0, 75.0, 100, 200 and 300 µg/L.
Correlation factors (r) were at least 0.9989
In sum, six series of measurements were performed for the determination of total dissolved chromium, manganese and nickel 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 chromium and manganese 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 11 the LODs and LOQs for chromium, manganese and nickel 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 100.848 mg/L to 101.862 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: Cr: 16.8 ± 0.32 µg/L, Fe: 62.7 ± 1.48 µg/L, Mn: 144 ± 0.75 µg/L; in GST: Cr: 23.7 ± 0.91 µg/L, Fe: 85.5 ± 6.34 µg/L, Mn: 170 ± 6.25 µg/L

Any other information on results incl. tables

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 0.9999
Limit of detection	Fe: 1.35 – 2.17 µg/L Mn: 6.58 – 13.8 µg/L
Limit of quantification	Fe: 4.51 – 7.27 µg/L Mn: 21.9 – 46.9 µg/L
Method blanks	GST: Method blanks 2h below LOD/LOQ; method blanks 24h three below LOD one above LOQ but at leat 12.4 fold lower than concentration in samples GMB: <LOD ALF: Method blanks above LOQ but at least 2.36 fold lower than concentrations in samples ASW: <LOD PBS: Method blanks 2h below LOQ, method blanks 24h above LOQ but at least 3.34 fold lower than concentrations in samples	Elevated concentrations of Fe in method blanks origin from the applied chemicals for preparation of different media
Accuracy samples	mean recovery for CRM TMDA-70: Fe: 103 ± 2.8 % (n = 6)	High concentration range (368 µg Fe/L)
Accuracy samples	mean recovery for CRM TM15.2: Fe: 101 ± 3.7 % (n = 6)	Low concentration range (25.8 µg Fe/L)
Accuracy mass balance	mean recovery for CRM TMDA-70: Mn: 100 ± 1.2 % (n = 8)	high concentration range (302 µg Mn/L)
Trueness measurement series in samples	mean recovery for recalibration standard : Fe: 102 ± 7.0 % (n = 6)	low concentration range (25 µg/L)
Trueness measurement series in samples	mean recovery for recalibration standard : Fe: 104 ± 4.3 % (n = 6)	mid concentration range (35 µg/L)
Trueness mass balance samples	mean recovery for recalibration standard : Mn: 99.2 ± 0.2 % (n = 5)	low concentration range (50 µg/L)
Trueness mass balance samples	mean recovery for recalibration standard : Mn: 100 ± 2.7 % (n = 8)	mid concentration range (100 µg/L)
Trueness mass balance samples	mean recovery for recalibration standard : Mn: 96.3 ± 0.8 % (n = 3)	high concentration range (250 µg/L)
Trueness measurement series in samples	Fortification of samples: Fe:97.6 – 107 %
Reproducibility sample measurement	mean recovery for CRM TMDA-70: Fe: 103 ± 2.8 % (n = 6)	High concentration range (368 µg Fe/L)
Reproducibility sample measurement	mean recovery for CRM TM15.2: Fe: 101 ± 3.7 % (n = 6)	Low concentration range (25.8 µg Fe/L)
Reproducibility sample measurement	mean recovery for CRM TMDA-70: Mn: 100 ± % (n = 8)	high concentration range (302 µg Mn/L)

 Method validation summary (ICP-MS):

validation parameter	results	Comment
Selectivity	Cr: similar in Helium and HiHelium mode Mn: similar in Helium and HiHelium mode	appropriate Isotope and gas mode were selected for interference free measurements
Linearity	applied calibration functions were linear	correlation coefficients ≥ 0.9989
Limit of detection	Cr: 0.01 – 0.33 µg/L Mn: 0.003 – 0.12 µg/L
Limit of quantification	Cr: 0.02 – 0.98 µg/L Mn: 0.01 – 0.36 µg/L
Method blanks	GST: Cr: Method blanks 2h three below LOD, one above LOQ; method blanks 24h three below LOD, one above LOQ but at least 136 fold lower than concentration in samples; Mn: Method blanks 2h below LOD, method blanks 24h two below LOD two above LOQ but at least 2150 fold lower than concentration in samples GMB: Cr: Method blanks 2h three below LOD one above LOQ but at least 28.0 fold lower than concentrations in samples; method blanks 24h two below LOD two below LOQ; Mn: Method blanks above LOQ but at least 4.92 fold lower than concentrations in samples ALF: Cr: <LOD/LOQ; Mn: <LOD/LOQ ASW: Cr: <LOD; Mn: Method blanks 2h below LOD, method blanks 24h three below LOD one above LOQ but at least 1850 fold lower than concentration in samples PBS: Cr: <LOD/LOQ; Mn: <LOD/LOQ	Elevated concentrations of Cr and Mn in method blanks origin from the applied chemicals for preparation of different media
Accuracy	mean recovery for CRM TMDA-70: Cr: 103 ± 7.2 % (n = 33) Mn: 102 ± 5.2 % (n = 33)	high concentration range (389 µg Cr/L; 302 µg Mn/L)
Accuracy	mean recovery for CRM TM15.2: Cr: 96.3 ± 8.6 % (n = 33) Mn: 98.8 ± 6.5 % (n = 33)	low concentration range (16.4 µg Cr/L; 18.1 µg Mn/L)
Trueness	mean recovery for recalibration standard: Cr: 100 ± 10 % (n = 31) Mn: 102 ± 5.2 % (n = 31)	low concentration range (1 µg/L)
Trueness	mean recovery for recalibration standard: Cr: 101 ± 6.7 % (n = 35) Mn: 101 ± 7.3 % (n = 35)	mid concentration range (10 µg/L)
Trueness	Fortification of samples: Cr: 89.8– 106 % Mn:96.8 – 104 %
Reproducibility	mean recovery for CRM TMDA-70: Cr: 103 ± 7.2 % (n = 33) Mn: 102 ± 5.2 % (n = 33)	high concentration range (389 µg Cr/L; 302 µg Mn/L)
Reproducibility	mean recovery for CRM TM15.2: Cr: 96.3 ± 8.6 % (n = 33) Mn: 98.8 ± 6.5 % (n = 33)	low concentration range (16.4 µg Cr/L; 18.1 µg Mn/L)

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 [%] normalizedfor measured background in method blank
ALF 2h	<LOD/LOQ	6.58 ± 0.25	6.58 ± 0.25	18755	0.04 ± <0.01
ALF 24h	<LOD/LOQ	16.8 ± 0.32	16.8 ± 0.32	18755	0.09 ± <0.01
ASW 2h	<LOD	1.92 ± 0.02	1.92 ± 0.02	18688	0.01 ± <0.01
ASW 24h	<LOD	3.24 ± 0.20	3.24 ± 0.20	18688	0.02 ± <0.01
GMB 2h	0.06	1.61 ± 0.40	1.55 ± 0.40	18666	0.01 ± <0.01
GMB 24h	<LOD/LOQ	1.86 ± 0.19	1.86 ± 0.19	18666	0.01± <0.01
GST 2h	0.07	9.01 ± 1.42	8.95 ± 1.42	18756	0.05 ± 0.01
GST 24h	0.06	23.8 ± 0.91	23.7 ± 0.91	18756	0.13 ± <0.01
PBS 2h	<LOD	1.24 ± 0.01	1.24 ± 0.01	18657	0.01 ± <0.01
PBS 24h	<LOD/LOQ	1.63 ± 0.18	1.63 ± 0.18	18657	0.01 ± <0.01

# (initial weight (e.g. 50 mg) * 18.47 § (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 27.00 % Cr as Cr₂O₃ => 68.42 % chromium in Cr₂O₃ => (27.00 % * 68.42 %)/ 100 % = 18.47 % Cr in test item

In five different artificial physiological media, between 0.01 and 0.13 % of chromium was dissolved from the test item IPC-2013-013 spinels, chromium iron manganese browndepending 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 [%] normalizedfor measured background in method blank
ALF 2h	21.1 ± 0.75	49.9 ± 0.06	28.7 ± 0.06	20593	0.14 ± <0.01
ALF 24h	20.7 ± 0.14	83.4 ± 1.48	62.7 ± 1.48	20593	0.30 ± 0.01
ASW 2h	<LOD	<LOD	-	20520	-
ASW 24h	<LOD	8.82	8.82	20520	0.04
GMB 2h	<LOD	<LOD	<LOD	20496	-
GMB 24h	<LOD	<LOD	<LOD/LOQ	20496	-
GST 2h	<LOD/LOQ	32.0 ± 6.11	32.0 ± 6.11	20594	0.16 ± 0.03
GST 24h	7.54	93.1 ± 6.34	85.5 ± 6.34	20594	0.42 ± 0.03
PBS 2h	<LOQ	<LOQ	-	20485	-
PBS 24h	5.98 ± 0.08	20.0	14.0	20485	0.07

# (initial weight (e.g. 50 mg) * 20.28 § (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 29.20 % Fe as Fe₂O₃ => 69.94 % iron in Fe₂O₃ => (29.20 % * 69.94 %)/ 100 % = 20.28 % Fe in test item.

In five different artificial physiological media, between 0.04 and 0.42 % of iron was dissolved from the test item IPC-2013-013 spinels, chromium iron manganese browndepending on solution parameters and test duration.

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

media and sample	total Mn ± SD in method blanks [µg/L]	total Mn ±SD in sample vessels [µg/L]	Mn ± SD in sample vessels with blank subtraction [µg/L]	calculated nominal Mn concentration in [µg/L]#	dissolved amount Mn in artificial media [%] normalizedfor measured background in method blank
ALF 2h	<LOD	81.7 ± 0.55	81.7 ± 0.55	27843	0.29 ± <0.01
ALF 24h	<LOD/LOQ	144 ± 0.75	144 ± 0.75	27843	0.52 ± <0.01
ASW 2h	<LOD	5.57 ± 2.32	5.57 ± 2.32	27744	0.02 ± 0.01
ASW 24h	0.02	37.4 ± 2.21	37.4 ± 2.21	27744	0.13 ± 0.01
GMB 2h	0.20 ± <0.01	1.83 ± 1.43	1.63 ± 1.43	27711	0.01 ± 0.01
GMB 24h	0.07 ± 0.01	0.36 ± 0.01	0.29 ± 0.01	27711	0.001 ± <0.001
GST 2h	<LOD	77.4 ± 8.43	77.4 ± 8.43	27844	0.28 ± 0.03
GST 24h	0.08 ± 0.05	170 ± 6.25	170 ± 6.25	27844	0.61 ± 0.02
PBS 2h	<LOD/LOQ	2.69 ± 0.59	2.69 ± 0.59	27697	0.01 ± <0.01
PBS 24h	<LOD/LOQ	11.3 ± 1.51	11.3 ± 1.51	27697	0.04 ± 0.01

# (initial weight (e.g. 50 mg) * 27.42 § (percentage manganese in test item)/ 100) * 2 (multiplication to calculate manganese amount in one litre --> 100 mg/L) = nominal manganese concentration in [mg/L]/1000 = nominal manganese concentration in [µg/L]

§ according to CoA 35.40 % Mn as MnO => 77.45 % manganese in MnO => (35.40 % * 77.45 %)/ 100 % = 27.42 % Mn in test item

In five different artificial physiological media, between 0.001 and 0.61 % of manganese was dissolved from the test item IPC-2013-013 spinels, chromium iron manganese brown depending on solution parameters and test duration.

Mass balance calculation

Total dissolved manganese 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 manganese mass balance

media	value for dissolved Mn after addition of aqua regia[mg]	nominal concentration [mg] #	recovery [%]
ALF 24h A	0.10	13.9	1.03
ALF 24h B	0.10	13.9	1.03
ASW 24h A	0.11	13.9	1.21
ASW 24h B	0.12	13.8	1.30
GMB 24h A	0.13	13.9	1.36
GMB 24h B	0.12	13.8	1.34
GST 24 A	0.14	13.9	1.48
GST 24h B	0.13	14.0	1.42
PBS 24h A	0.12	13.9	1.31
PBS 24h B	0.12	13.8	1.33

# nominal concentration Mn = 35.40 % as MnO in test item = 77.45 % Mn in MnO => 13.708 mg Mn in 50 mg test item => 13.708 * initial weight / 50 mg

Applicant's summary and conclusion

Conclusions:

The bioaccessibility of spinels, chromium iron manganese brown has been investigated experimentally in vitro by simulating dissolution under physiological conditions considered to mimic the most relevant exposure routes (oral, dermal and inhalation). Dissolved Cr, Mn and Fe concentrations were below 23.7 µg/L, 170 µg/L, and 85.5 µg/L respectively, even at the highest loading of 0.1 g/L, referring to a solubility of 0.024 %, 0.17 %, and 0.086 %, respectively, the pigment is considered biologically inert.

Executive summary:

The bioaccessibility of spinels, chromium iron manganes brown 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 five different artificial physiological media dissolved chromium concentrations were between 0.01 and 0.13 %, dissolved iron concentrations were between < LoD and 0.42 % and dissolved manganese concentrations were between 0.01 and 0.61 %, based on contained elements.

Therefore, spinels, chromium iron manganes brown may reasonably be considered not bioaccessible.