Triiron tetraoxide

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-standard animal species; non-GLP

Reason / purpose for cross-reference:

reference to same study

Objective of study:

toxicokinetics

Principles of method if other than guideline:

Commercial magnetite of an MMAD of ~3 µm was selected. Measurement indicated an MMAD of 3.4 µm. The purity was 98%. The material was neutron-activated to produce an activity of 125 µCi/g of 59Fe Number of animals: 4 Duration of Exposure: 4 h
Concentration: 30- 85- 151- 161 mg/m³ corresponding to lung burdens of 21- 64- 75- 110 mg Clearance
was measured radiometrically 1-2-4-7 days after exposure, then weekly for 12 weeks and bimonthly until ~210 days post
exposure. Activity at 24 h post dose was set as 100% (= no clearance from the alveoli)

GLP compliance:

no

Radiolabelling:

yes

Species:

other: donkey

Strain:

not specified

Sex:

male/female

Route of administration:

inhalation

Vehicle:

not specified

Duration and frequency of treatment / exposure:

4 hour(s)

Remarks:

Doses / Concentrations:
Males: 30, 85, 151, 161 mg/m³ Females: 30, 85, 151, 161 mg/m³

No. of animals per sex per dose / concentration:

Males: 2 Females: 2

Control animals:

no

Preliminary studies:

no data

Details on absorption:

no data

Details on distribution in tissues:

no data

Details on excretion:

see toxicokinetik parameters

Toxicokinetic parameters:

half-life 2nd: 42 - 173 days

Toxicokinetic parameters:

half-life 1st: 15 - 22 days

Metabolites identified:

no

Details on metabolites:

inert particles - no metabolites identified (not examined)

Magnetite showed a two-phase lung clearance with halflives of about 3 
weeks in the first phase and several months in the second phase

Executive summary:

Commercial magnetite of an MMAD of ~3 µm was selected . Measurement indicated an MMAD of 3.4 µm.

The purity was 98%. The material was neutron-activated to produce an activity of 125 µCi/g of 59Fe Number

of animals: 4 Duration of Exposure: 4 h Concentration: 30- 85- 151- 161 mg/m³ corresponding to lung burdens

of 21- 64- 75- 110 mg Clearance was measured radiometrically 1-2-4-7 days after exposure, then weekly for

exposure. Activity at 24 h post dose was set as 100% (= no clearance from the alveoli)

As a result Magnetite showed a two-phase lung clearance with halflives of about 3 weeks in the  first phase and several months in the second phase

Reference 2

Endpoint:

basic toxicokinetics in vitro / ex vivo

Remarks:

Bioaccessibility

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-02-26 to 2018-09-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Objective of study:

bioaccessibility (or bioavailability)

Qualifier:

according 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

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 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 conducted 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. The test media were artificial physiological media: gastric fluid (GST), phosphate-buffered saline (PBS), artificial lysosomal fluid (ALF) and Gamble's solution (GMB)

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2016-05-31

Radiolabelling:

no

Species:

other: in vitro (simulated human body fluids)

Details on test animals or test system and environmental conditions:

Test principle in brief:
- four different artificial physiological media,
- single loading of test substance of ~100 mg/L,
- GST and PBS media: samples taken after 2 and 24 hours agitation (100 rpm) at 37 ± 2 °C
- GMB and ALF media: samples taken after 2, 24 and 168 hours agitation (100 rpm) at 37 ± 2 °C

- two additional method blanks per medium, measurement (ICP-OES) of dissolved Fe concentrations after filtration and centrifugal filtration.
- the study was performed in triplicate

The aim of this test was to assess the dissolution of triiron tetraoxide (Ferroxide Black 86) in four artificial physiological media: Phosphate buffered saline (PBS, pH 7.2-7.4), Artificial gastric fluid (GST, 1.5-1.6), artificial lysosomal fluid (ALF) and Gamble’s solution (GMB). The test media were selected to simulate relevant human-chemical interactions (as far as practical), e.g. a substance entering the human body by ingestion into the gastro-intestinal tract (GST) or via the respiratory system (ALF).

Duration and frequency of treatment / exposure:

Iron concentrations in GST and PBS were determined after 2 and 24 h whereas iron concentrations in GMB and ALF media were assessed after 2, 24 and 168 hours of incubation.

Dose / conc.:

100 other: mg of test item/L artificial media

Details on study design:

Test setup
Three replicate flasks (500 mL glass flasks) per test medium (PBS, GST) were prepared with a loading of ~ 100 mg/L. The test item was weighed into flasks, adjusted to volume with the respective artificial physiological medium and agitated at 100 rpm at 37°C ± 2°C. Two control blank replicates (same procedure) per test medium were also prepared.
Three replicates containing the test item and two method blanks per artificial medium were tested. All solutions were sampled after 2 and 24 h whereas GMB and ALF media were also sampled after 168h to measure total dissolved Fe concentrations (ICP-OES) after 0.2 µm filtration (Syringe Filter w / 0.2 μm, polyethersulfon membrane, DIA Nielsen, Dueren, Germany) and centrifugal filtration (i.e. 0.2 μm filtration and 3kDa centrifugal filtration, Sartorius, Göttingen, Germany). In addition, temperature, pH and observations, including the appearance of the solution (including colour, turbidity and particle film on the surface) were recorded.


Sample fortification:
In addition, samples of the artificial physiological media were fortified with a known amount of iron (by standard addition of commercial standards) to determine the standard recovery. For detailed information please refer to "Any other information on materials and methods incl. tables".

Mass balance:
After the test, aqua regia (3 : 1 mixture of concentrated hydrochloric and nitric acid) was added to the vessels containing the test item to reach a final volume of 500 mL, i.e. 120 mL aqua regia were added to approximately 380 mL GST or PBS medium, 180 mL aqua regia were added to approximately 320 mL ALF or GMB medium. From these solutions, 50 mL were taken after 3 - 14 days of “digestion” for mass balance determination.
The filters (Syringe Filter w / 0.2 μm, polyethersulfon membrane, DIA Nielsen, Dueren, Germany) used for sampling were extensively rinsed with a known volume of aqua regia (ca. 2.5 mL). The added aqua regia was let to drop slowly through the filters and was collected in a clean vial. This procedure was repeated with every syringe and filter used during the study. After collection, the volume was filled up to exactly 10 mL (for the media GST and PBS) and up to 15 mL (for the ALF and GMB media) with aqua regia. Afterwards the concentration of iron in the “filtrated” aqua regia was determined and considered for the determination of the mass balances.

Reagents:
Purified water (resistivity > 18 MΩ·cm, Pure Lab Ultra water purification system from ELGA LabWater, Celle, Germany)
Nitric acid - “Supra” quality (ROTIPURAN® supplied by Roth, Karlsruhe, Germany).
Hydrochloric acid – “Baker-instra-analyzed-plus” quality (J.T. Baker, Griesheim, Germany).
Sodiumhydroxide – pro Analysis quality (Chemsolute, Th. Geyer, Renningen, Germany)
MgCl2 x 6H2O (p.A., Merck, Darmstadt, Germany)
NaCl (p.A., Chemsolute, Renningen, Germany + Merck, Darmstadt, Germany (new GMB medium))
KCl (p.A., Chemsolute, Renningen, Germany + Merck, Darmstadt, Germany (new GMB medium))
Na2HPO4 (p.A. Merck, Darmstadt, Germany)
Na2SO4 (p.A. Merck, Darmstadt, Germany)
CaCl2 x 2H2O (p.A. Merck, Darmstadt, Germany)
NaAcetate (suprapur Merck, Darmstadt, Germany)
NaHCO3 (p.A. Merck, Darmstadt, Germany)
NaOH (p.A., Chemsolute, Renningen, Germany)
Citric acid anhydrous (p.A., Roth, Karlsruhe, Germany)
Glycine (p.A., Merck, Darmstadt, Germany)
Na3Citrate x 2H2O (p.A., Merck, Darmstadt, Germany)
Na2Tartrate x 2H2O (p.A., Merck, Darmstadt, Germany)
NaLactate (98+% Sigma Aldrich, Munich, Germany)
NaPyruvate (p.A., Applichem, Darmstadt, Germany)
KH2PO4 (p.A., Merck, Darmstadt, Germany)
Urea (pure, Applichem, Darmstadt, Germany)
Lactic acid (purum, Fluka, Munich, Germany)
HCl 30% (instra-analyzed, plus J.T. Baker, Griesheim, Germany)


METAL ANALYSIS
- Standards for metal analysis: A commercially available single element standard was used as iron standard (Merck Certipur Iron ICP standard 1000 mg/L lot no. HC68868126; Darmstadt, Germany) to prepare an appropriate stock solution and subsequently calibration solutions for ICP-OES measurements
- Certified reference materials: As quality control standards, certified aqueous reference material TM-DWS.3 (lot no. 0916) and TMDA-70.2 (lot no. 0916 and 0917) obtained from Environment Canada and a multielement standard (Merck Certipur IV ICP standard 1000 mg/L lot no. HC54938555 and HC73962555; Darmstadt, Germany) were analysed for total dissolved iron by ICP-OES.

Instrumental and analytical set-up for the ICP-OES instrument:
Agilent 720, Agilent Technologies, Waldbronn, Germany
Nebulizer: Sea spray nebulizer, from Glass Expansion
Spray chamber: Iso Mist with Twister Helix from Glass Expansion
Plasma stabilization time: at least 30 min before start of the measurements
Plasma gas flow: 15.0 L/min
Additional gas flow: 1.50 L/min
Carrier gas flow: 0.75 L/min
RF power: 1200W
Stabilization time of sample: 15 sec
Repetition time (three internal measurements per sample): 30 sec
Wavelengths: Fe: 238.204 nm, 240.489 nm, 241.052 nm, 258.588 nm and 259.940 nm

- Correlation coefficients (r) for the wavelengths used for evaluation of data were at least >0.999603

The applied LOD/LOQ calculations for the Agilent 720 ICP-OES:
LOD: 3 * standard deviation of calibration blank/slope of the calibration
LOQ: 3 * LOD
The resulting LODs/LOQs are reported in "Any other information on results incl. tables"

Details on dosing and sampling:

Loading:
Detailed loadings of the test vessels are given in "Any other information on materials and methods incl. tables".

Type:

other: Bioaccessibility ALF, 2h @ 37°C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

742 µg Fe/L (dissolved)

Type:

other: Bioaccessibility ALF, 24h @ 37°C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

30824 µg Fe/L (dissolved)

Type:

other: Bioaccessibility ALF, 168h @ 37°C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

75367 µg Fe/L (dissolved)

Type:

other: Bioaccessibility GST, 2h @ 37°C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

296 µg Fe/L (dissolved)

Type:

other: Bioaccessibility GST, 24h @ 37°C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

1186 µg Fe/L (dissolved)

Type:

other: Bioaccessibility GMB, 2h @ 37°C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

0.106 µg Fe/L

Type:

other: Bioaccessibility GMB, 24h @ 37°C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

< LOD (0.19 µg Fe/L)

Type:

other: Bioaccessibility GMB, 168h @ 37°C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

2.16 µg Fe/L (dissolved)

Type:

other: Bioaccessibility PBS, 2h @ 37°C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

Type:

other: Bioaccessibility PBS, 24h @ 37°C (100 mg/L loading, 0.2 µm + 3 kDa filtration for phase separation)

Results:

< LOD/LOQ (2.28 µg Fe/L) (dissolved)

Bioaccessibility (or Bioavailability) testing results:

Please refer to "any other information on results incl. tables" below.

Iron concentrations in simulated artificial body fluids:

The bioaccessibility of Ferroxide Black 86 was determined in vitro by simulating dissolution under physiological conditions considered to mimic artificial body fluids with a loading of 100 mg test item/L. Dissolved iron concentrations were operationally defined as the dissolved Fe fraction after 0.2 µm filtration and centrifugal filtration (~2.1 nm), see Table 3. With a maximum mean released fraction of ~7.5% after 168 h, dissolution of Ferroxide Black 86 was highest in artificial lysosomal fluid (ALF).

In addition, dissolved/dispersed mean iron concentrations (operationally defined as the dissolved Fe fraction after 0.2 µm filtration) are summarized in table 4.

Table 3 is provided as an Attachment to this entry.

Table 4 is provided as an Attachment to this entry.

 

Mass balance

Total mass recoveries were determined by aqua regia digestion for each test item containing vessel at the end of the experiment. Regarding Ferroxide Black 86, mass recoveries in all media investigated (GST, GMB, ALF, PBS) were > 95.0%.

 

Solution pH -GST

 

blank vessels

sample name	target pH	pH prior to the test	pH after 2h	pH after 24 h
GSTblankvessel1	1.5–1.6	1.55	1.57	1.59
GSTblankvessel2	1.5–1.6	1.54	1.58	1.58

 

Ferroxide Black 86

sample name	target pH	pH prior to the test	pH after 2h		pH after 24h
GSTvessel10	1.5–1.6	1.55		1.61	1.61
GSTvessel11	1.5–1.6	1.55		1.60	1.65
GSTvessel12	1.5–1.6	1.54		1.61	1.62

 

Solution pH - PBS

 

blank vessels

samplename	targetpH	pHpriortothetest	pHafter2h	pHafter24h
PBSblankvessel1	7.2–7.4	7.30	7.30	7.33
PBSblankvessel2	7.2–7.4	7.30	7.33	7.33

 

Ferroxide Black 86

samplename	targetpH	pHpriortothetest	pHafter2h	pHafter24h
PBSvessel10	7.2–7.4	7.30	7.34	7.37
PBSvessel11	7.2–7.4	7.30	7.33	7.35
PBS vessel 12	7.2–7.4	7.29	7.34

 

Solution pH - GMB

 

blank vessels

samplename	targetpH	pHpriortothetest	pHafter2h	pHafter24h	pHafter7d
GMBblankvessel1	7.4	7.46	8.10	8.68	9.09
GMBblankvessel2	7.4	7.46	8.14	8.74	9.09

 

 

Ferroxide Black 86

samplename	targetpH	pHpriortothetest	pHafter2h	pHafter24h	pHafter7d
GMB vessel 10	7.4	7.45	8.21	8.83	9.17
GMB vessel 11	7.4	7.44	8.17	8.85	9.17
GMB vessel 12	7.4	7.45	8.18	8.88	9.18

 

 

Solution pH - ALF

 

blank vessels

samplename	targetpH	pHpriortothetest	pHafter2h	pHafter24h	pHafter7d
ALFblankvessel1	4.5	4.56	4.57	4.62	4.62
ALFblankvessel2	4.5	4.56	4.59	4.62	4.62

 

 

Sicovit Red 30E172

samplename	targetpH	pHpriortothetest	pHafter2h	pHafter24h	pHafter7d
ALF vessel 10	4.5	4.55	4.60	4.65	4.64
ALF vessel 11	4.5	4.54	4.59	4.62	4.67
ALF vessel 12	4.5	4.56	4.62	4.65	4.67

 

 

Test temperature:

With 37 °C ± 2 °C, the temperature was stable during the test for all solutions

 

 

Method validation summary (ICP-OES)

Limits of detection (LODs), limits of quantification (LOQs) and correlation coefficients (r)

Limits of detection (LOD) within all measurement series: < 1.24 µg Fe/ L.

Limits of quantification (LOQ) within all measurement series: < 3.73 µg Fe/ L.

Correlation coefficients (r) within all measurement series: >0.999603

 

GST

Mean recovery of fortified samples (n = 20): 98.6 - 104 %

Mean recoveries for certified reference materialTM-DWS.3andTMDA-70.2(concentration range 22.4 – 75.4 µg Fe / L, n = 24): 103 -105 %

Mean recoveries for quality control standard (concentration range 50 -500 µg Fe/ L, n = 24): 98.2 - 105 %

Mean recoveries for internal standard (concentration range 10 -300 µg / L, n = 24): 98.4 - 100 %

 

PBS

Mean recovery of fortified samples (n = 32): 90.5 - 108 %

Mean recoveries for certified reference materialTM-DWS.3andTMDA-70.2(concentration range 22.4 – 75.4 µg Fe / L, n = 20): 98.0-101 %

Mean recoveries for quality control standard (concentration range 5 -50 µg Fe/ L, n = 30): 97.0 – 99.9 %

Mean recoveries for internal standard (concentration range 10 -100 µg / L, n = 30): 97.5 – 99.8 %

 

GMB

Mean recovery of fortified samples (n = 48): 92.2 - 128 %

Mean recoveries for certified reference materialTM-DWS.3andTMDA-70.2(concentration range 22.4 – 75.4 µg Fe / L, n = 59): 95.5-107 %

Mean recoveries for quality control standard (concentration range 5 -50 µg Fe/ L, n = 60): 95.5 – 104 %

Mean recoveries for internal standard (concentration range 10 -100 µg / L, n = 60): 97.0 – 102 %

 

ALF

Mean recovery of fortified samples (n = 24): 85.5 - 106 %

Mean recoveries for certified reference materialTM-DWS.3andTMDA-70.2(concentration range 22.4 – 75.4 µg Fe / L, n = 48): 93.6-103 %

Mean recoveries for quality control standard (concentration range 5 -200 µg Fe/ L, n = 48): 96.1-100 %

Mean recoveries for internal standard (concentration range 10 -100 µg / L, n = 48): 95.0 – 106 %

 

Method validation – mass balance measurements

Mean recovery of fortified samples (n = 27): 89.4 - 106 %

Mean recoveries for certified reference materialTM-DWS.3andTMDA-70.2(concentration range 22.4 – 75.4 µg Fe / L, n = 30): 100-109 %

Mean recoveries for quality control standard (concentration range 250 -600 µg Fe/ L, n = 30): 100-103 %

Mean recoveries for internal standard (concentration range 100 -400 µg / L, n = 30): 100 – 106 %

Conclusions:

The bioaccessibility of Ferroxide Black 86 was determined in vitro by simulating dissolution under physiological conditions considered to mimic artificial body fluids with a loading of 100 mg test item/L. After 2 and 24 h in phosphate buffered saline (PBS, pH 7.2-7.4) solution, dissolved iron concentrations (operationally defined as the dissolved Fe fraction after 0.2 µm filtration and centrifugal filtration (~2.1 nm)) were below the LOD (<0.761 µg/L). With mean concentrations of 0.106 µg Fe/L after 2 h and 2.16 µg Fe/L after 168 h as well as a negative value (after subtraction of background) after 24h, dissolved iron concentrations in Gamble´s solution (GMB, pH 7.4) were also very low. In artificial gastric fluid (GST, pH 1.5-1.6), 296 µg Fe/L and 1.186 µg Fe/L were detected in the dissolved phase after 2 and 24 h, respectively. Mean iron concentrations were highest in artificial lysosomal fluid (ALF, pH 4.5): 742 µg/L, 30,824 µg/L and 75,367 µg/L of iron were found in the dissolved phase after 2, 24 and 168 h. Therefore, the test item is fully soluble in artificial lysosomal fluid (ALF) after 168 h.

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-09-11 to 2019-06-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

toxicokinetics

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

2010-07-22

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2017-05-08

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored in tightly-closed, original container, in a cool and dry place.

Radiolabelling:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat is a commonly used rodent species for toxicity studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Research, Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 59 to 60 days
- Weight at study initiation: males: 262 g to 340 g; females: 200 g to 272 g
- Housing: kept in groups of up to 3 animals (same sex) in MAKROLON cages (type IV) with a basal surface of approximately 55 cm × 33 cm and a height of approximately 20 cm; bedding material: granulated textured wood
- Diet (ad libitum): Commercial diet, ssniff® R/M-H V1534
- Water (ad libitum): drinking water
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- Relative humidity: 55% ± 10% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

other: Ferroxide black 86: oral (gavage); reference item: intravenously injected

Vehicle:

other: Ferroxide black 86: 0.5 % aqueous hydroxypropylmethylcellulose gel; reference item: 0.9 % NaCl solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
1) Ferroxide black 86:
The test items were suspended or dissolved in the vehicle to the appropriate concentration freshly on the administration day and were administered orally by gavage at a constant volume (adminsitration volume: 10 mL/kg bw). The application formulations were continuously agitated by stirring throughout the entire administration procedure.

2) Reference item (Iron (III) citrate tribasic monohydrate; Fe content: 21.2%)
Prior to administration, the reference item and the appropriate vehicle were heated to 70°C and stirred at 50°C for approx. 3 hours until the reference item was completely dissolved. This clear solution was maintained at room temperature until administration. The status as clear solution was monitored and recorded upon administration. Immediately after formulation preparation for the females, the formulations were protected from light by transferring the formulation into brown containers or wrapping in aluminium foil.

The amounts of the test and reference items were adjusted to the animal's current body weight on the administration day.

Administration volume (oral administration / intravenous administration): 10 mL/kg bw/day

Injection speed (intravenous adminsitration): dose per approx. 15 seconds

Duration and frequency of treatment / exposure:

single administration

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose / concentration:

5 males / 5 females

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

none

Details on study design:

- Dose selection rationale: the dose levels for this study were selected after consultation with the sponsor based on available toxicity and bioavailability data (as far as available):

1) Reference item (Iron (III) citrate tribasic monohydrate; Fe content: 21.2%):
The oral LD50 value for iron citrate monohydrate was stated as being >2000 mg/kg bw; the oral bioavailability of soluble Fe substances are given in the public domain with 1 to 26% (Fe).

For the test item oral dosing of 1000 mg/kg bw, a very low relative bioavailability was assumed (<1%), considering the very low water solubility and bioacessibility in gastric juice. Since the four iron oxide test items have Fe-contents of approx. 70%, the dose of the reference substance should be adjusted accordingly. Given a test item dose of 1000 mg/kg b.w. (corresponding to 700 mg Fe/kg bw), then 1% of this dose would correspond to 7 mg Fe/kg bw (or 36.8 mg/kg bw iron citrate). Correcting for approx. 20% oral bioavailability of soluble iron substances, this yields a dose for the reference item of 7.4 mg iron citrate/kg bw to be given by intravenous injection.

2) Ferroxide black 86:
The test item oral doses of 1000 mg/kg bw correspond to the limit dose used in a separate 90-day oral toxicity study, which was considered the maximum feasible dose. This dose was also selected in view of the anticipated low bioavailability and the requirements of analytical sensitivity of the analytical method for iron in plasma.

3) Vehicle control group:
In view of the long established circadian variation of plasma iron levels (Lynch et al, 1973)*, a vehicle control group was sampled for blood plasma over a period of 24 hours at identical sampling time points and intervals as the dosed groups.

*Reference:
Lynch et al (1973): Circadian Variation in Plasma Iron Concentration and Reticuloendothelial Iron Release in the Rat, Clinical Science and Molecular Medicine (1973) 45, 331-336.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled (delete / add / specify): urine, faeces, blood, plasma, serum or other tissues, cage washes, bile
- Time and frequency of sampling: blood was collected 0 (predose), 0.5, 1, 2, 4, 8, 12, 24, 48 (test item and reference item only), and 72 hours (test item and reference item only) after administration. The whole blood samples were cooled using an IsoTherm-Rack system until centrifugation. Immediately after centrifugation, the isolated plasma was frozen at -20°C ± 10 % and stored at this temperature until analysis.

Pharmacokinetic evaluation of plasma data was performed and a non-compartment model was employed. The following parameters were determined, if possible:
AUC0-∞ = extrapolated area from zero to infinity
AUC0-t last = extrapolated area from time zero to the last quantifiable plasma concentration (i.e. >lower limit of quantification, LLOQ)
Kel = elimination rate constant
t1/2 = elimination half-life

Cmax values were the highest measured plasma concentrations and tmax values were the time points of highest plasma concentrations.

Elimination rate constants (Kel) and plasma elimination half-lives (t½) were calculated by linear regression analysis of the log/linear portion of the individual plasma concentration-time curves (c = concentration, t = time).

Area under the curve (AUC) values were calculated using the linear trapezoidal method and extrapolated to infinite time by dividing the last measurable plasma concentration by the elimination rate constant. Plasma concentrations at time zero were taken to be those at the first blood sampling time.

Furthermore, the AUC0-t last was calculated according to the linear trapezoidal rule. Values below the limit of quantification (LOQ) were excluded from calculation.

In addition, the bioavailability was calculated for the mixture.

For plasma, a pre-treatment by a microwave digestion with HNO3 was necessary to digest the proteins in plasma. Afterwards iron in digested samples was measured by ICP-OES.

OBSERVATIONS
- clinical signs: before and after dosing as well as regularly throughout the working day (7.30 a.m. to 4.30 p.m.) and on Saturdays and Sundays (8.00 a.m. to 12.00 noon; final check at approx. 4.00 p.m).
Special attention was paid to the local tolerance at the injection site(s).
- mortality/morbund: early in the morning and again in the afternoon of each working day as well as on Saturdays and Sundays (final check at approx. 4.00 p.m).
- body weight: at the time of group allocation, before dosing for dose adjustment and on test day 4 before the last blood sampling.

ADMINISTRATION FORMULATION ALANYSIS:
For each test item, that was mixed with the vehicle and the reference substance, tests by appropriate analytical methods were conducted to determine the concentration and stability of the test item in the formulations. For the analysis of the application formulations, one sample of exactly 10 mL from each dosing suspension (test items) or dosing solution (reference item) was taken at the start of the administration (test day 1 of the female animals) and frozen until analysis.

Application solutions of the iron oxide was measured after addition of aqua regia to the samples and after an incubation time for at least four days by ICP-OES. After this measurement the remaining precipitation (only iron oxide application solution) were digested by a microwave procedure and measured by ICP-OES.

ANALYTIC OF REFERENCE ITEM:
The iron content of the reference item (Iron (III) citrate tribasic monohydrate; Fe content: 21.2%) was determined using ICP-OES.

Statistics:

The test item group was compared to the reference group. The following statistical method was used:
- Student's t-test (body weight (at p≤0.05 and p≤0.01; limits used p = 0.05 approx. t = 2.306 p = 0.01 approx. t = 3.355 (for 8 degrees of freedom))

Preliminary studies:

none

Details on absorption:

not specified

Details on distribution in tissues:

not specified

Details on excretion:

not specified

Toxicokinetic parameters:

other: bioavailability

Remarks:

An absolute bioavailability of 0.23%/0.21% (m/f) for Ferroxide Black was calculated for iron following oral administration compared to intravenous administration.

Toxicokinetic parameters:

other:

Remarks:

It should be noted that this evaluation was done on the substance-specific data without consideration of the vehicle control.

Toxicokinetic parameters:

other:

Remarks:

The plasma iron level of the dosed group falls practically within the boundaries of the vehicle control group which reflects long established daily circadian variation of plasma iron levels.

Toxicokinetic parameters:

other:

Remarks:

The calculated absolute bioavailability derived by the pharmacokinetic analysis can therefore be seen as conservative overestimates, thus leading to the conclusion that the bioavailability of iron from the tested oxide is similarly minimal to negligible.

Metabolites identified:

not specified

Details on metabolites:

not specified

Bioaccessibility (or Bioavailability) testing results:

An absolute bioavailability of 0.23%/0.21% (m/f) for Ferroxide Black was calculated for iron following oral administration compared to intravenous administration. However, it should be noted that this evaluation was done on the substance-specific data without consideration of the vehicle control.
The plasma iron level of the dosed group falls practically within the boundaries of the vehicle control group which reflects long established daily circadian variation of plasma iron levels. Hence, the calculated absolute bioavailability derived by the pharmacokinetic analysis can therefore be seen as conservative overestimates, thus leading to the conclusion that the bioavailability of iron from the tested oxide is similarly minimal to negligible.

Please also refer for results to the field "Attached background information" below.

LOCAL TOLERANCE (REFERENCE ITEM; INTRAVENOUS ADMINISTRATION):

No signs of local intolerance reactions were noted at the injection sites of any male or female animal treated intravenously with 7.4 mg/kg Iron(III) citrate (reference item).

CLINICAL SIGNS, MORTALITY, AND BODY WEIGHT:

1) Ferroxide black 86:

- none of the animals died or had to be sacrificed prematurely. No signs of morbidity were noted.

- no signs of test item-related behavioural changes or abnormalities in the external appearance were noted for any male or female animal following single oral administration of Ferroxide black 86 at a dose level of 1000 mg/kg bw.

- discolouration of the faeces (black) was noted for all animals following single oral administration of the test item. The discolouration is however not considered a toxic effect, instead considered to be merely excretion of the respective test item.

- no test item-related changes were noted in body weight for any animal following single oral administration of Ferroxide black 86 at a dose level of 1000 mg/kg bw. No statistically significant differences were noted comparing the test item-treated group with the control group. The body weights were within the normal biological range of animals of this age and strain.

2) Reference item (iron (III) citrate tribasic monohydrate):

- none of the animals died or had to be sacrificed prematurely. No signs of morbidity were noted.

- signs of toxicity were noted for the male animals treated intravenously with the reference item Iron(III) citrate tribasic monohydrate with 7.4 mg/kg bw.. Reduced motility was noted for four male animals starting approx. 0-5 min p.a., lasting approx. 5-20 min. For the remaining male animal reduced motility was observed slightly longer with approx. 20-60 min accompanied with being in prone position. The female animals treated intravenously with the reference item did not reveal any abnormalities.

3) Vehicle control group:

- no signs of behavioural changes or abnormalities in the external appearance for any male or female animal following single oral administration of 0.5% aqueous hydroxypropylmethyl-cellulose gel were noted.

PHARMACOKINETIC EVALUATION

1) Reference item (iron (III) citrate tribasic monohydrate):

Cmax-levels in plasma of 6.28 μg Fe/g and 5.81 μg Fe/g were noted 0 to 1 hour (tmax as range m/f) after intravenous administration of 7.4 mg Iron(III) citrate/kg bw for the male and female rats on test day 1, respectively.

2) Ferroxide black 86:

Cmax-levels in plasma of 4.56 μg Fe/g and 4.68 μg Fe/g were noted 0 to 72 hours (tmax as range m/f) after oral administration of 1000 mg Ferroxide Black/kg bw for the male and female rats on test day 1, respectively.

TEST ITEM FORMULATION ANALYSIS:

The results of the analysis showed that the test item-formulation was correctly prepared. The actual concentration of iron in the formulation solution ranged from 92% to 96% and was well within the expected range of 90% to 110% of the theoretical concentration.

ANALYTIC OF REFERENCE ITEM:

1) Reference item (iron (III) citrate tribasic monohydrate):

The total iron content of the reference substance iron(III) citrate tribasic monohydrate determined after digestion by ICP-OES amounts to 21.2 % [w/w]. Measured iron, citrate and water contents of 21.2, 67.83 and 10.2 all in % [w/w], respectively, add up to 99.23 % [w/w]. Impurities were quantified in total with 0.19 % [w/w].

The iron content of 18.7% reported by the material supplier reflects only Fe(II) because of the iodometric titration employed. Considering measurement uncertainties, the reference substance iron(III) citrate tribasic monohydrate is considered adequately characterised, and the value of 21.2% total iron content should be taken forward.

Conclusions:

An absolute bioavailability of 0.23%/0.21% (m/f) for Ferroxide Black was calculated for iron following oral administration compared to intravenous administration. However, it should be noted that this evaluation was done on the substance-specific data without consideration of the vehicle control.
The plasma iron level of the dosed group falls practically within the boundaries of the vehicle control group which reflects long established daily circadian variation of plasma iron levels. Hence, the calculated absolute bioavailability derived by the pharmacokinetic analysis can therefore be seen as conservative overestimates, thus leading to the conclusion that the bioavailability of iron from the tested oxide is similarly minimal to negligible.

Description of key information

The dissolved Fe concentrations from triiron tetraoxide under simulated physiological conditions were highest in artificial lysosomal fluid (ALF, pH 4.5). 742 µg/L, 30,824 µg/L and 75,367 µg/L of iron were found in the dissolved phase after 2, 24 and 168 h (Ferroxide Black 86). With a maximum mean released fraction after 168 h, in artificial lysosomal fluid (ALF). This test item is fully soluble in ALF medium

An absolute bioavailability of 0.23%/0.21% (m/f) for Ferroxide Black 86 was calculated for iron following oral administration compared to intravenous administration. However, it should be noted that this evaluation was done on the substance-specific data without consideration of the vehicle control.

The plasma iron level of the dosed group falls practically within the boundaries of the vehicle control group which reflects long established daily circadian variation of plasma iron levels. Hence, the calculated absolute bioavailability derived by the pharmacokinetic analysis can therefore be seen as conservative overestimates, thus leading to the conclusion that the bioavailability of iron from the tested oxide is similarly minimal to negligible.

In the study by Schlesinger (1981) donkeys were exposed to commercial magnetite of an MMAD of ~3 µm for 4 h. Concentration: 30- 85- 151- 161 mg/m³ corresponding to lung burdens of 21- 64- 75- 110 mg.

Clearance was measured radiometrically 1-2-4-7 days after exposure, then weekly for 12 weeks and bimonthly until ~210 days post exposure. Activity at 24 h post dose was set as 100% (= no clearance from the alveoli). Magnetite showed a two-phase lung clearance with half-lives of about 3 weeks in the first phase and several months in the second phase.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

As demonstrated in the category approach justification, in regard to structure, physico-chemical properties, environmental fate characteristics, ecotoxicity and toxicity, the grouping of Fe2O3 (diiron trioxide), Fe3O4 (triiron tetraoxide), FeOOH (iron hydroxide oxide), (Fe,Mn)2O3 (iron manganese trioxide), (Fe,Mn)3O4 (manganese ferrite), and ZnFe2O4 (zinc ferrite) in the "Iron Oxides Category" is justified.

In the updated category approach justification , the category was extended to nano- and/or powder material. Comprehensive and sufficient data are available to conclude that nano- and micro-sized category members behave similar and no further testing is necessary.

Notably, from the results of repeated dose inhalation studies with FeOOH, Fe2O3 or Fe3O4 any size dependent increased translocation of iron outside the lung did not occur. Therefore no systemic toxicity for nano- and powder material of the iron oxides exists.

These repeated dose toxicity studies demonstrated, that the occupational limit value for nano- and powder material is identical.