Diiron trioxide

Basic toxicokinetics

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Objective of study:

toxicokinetics

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Diiron trioxide

EC Number:

215-168-2

EC Name:

Diiron trioxide

Cas Number:

1309-37-1

Molecular formula:

Fe2O3

IUPAC Name:

diiron(3+) trioxidandiide

Test material form:

solid: particulate/powder

Details on test material:

Appearance: red powder, odourless

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, kept dry and stored in airtight closed containers.

Radiolabelling:

no

Test animals

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

Administration / exposure

Route of administration:

other: Sicovit Red 30 E172: oral (gavage); reference item: intravenously injected

Vehicle:

other: Sicovit Red 30 E172: 0.5 % aqueous hydroxypropylmethylcellulose gel; reference item: 0.9 % NaCl solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
1) Sicovit Red 30 E172:
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

Doses / concentrations

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) Sicovit Red 30 E172:
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: plasma
- 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))

Results and discussion

Preliminary studies:

none

Toxicokinetic / pharmacokinetic studies

Details on absorption:

not specified

Details on distribution in tissues:

not specified

Details on excretion:

not specified

Toxicokinetic parameters

Toxicokinetic parameters 1

Toxicokinetic parameters:

other: bioavailability

Remarks:

An absolute bioavailability of 0.22%/0.23% (m/f) for Sicivit Red was calculated for Fe following oral administration compared to intravenous administration.

Toxicokinetic parameters 2

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 3

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 4

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.

Metabolite characterisation studies

Metabolites identified:

not specified

Details on metabolites:

not specified

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:

An absolute bioavailability of 0.22%/0.23% (m/f) for Sicivit Red 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.

Any other information on results incl. tables

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) Sicovit Red 30 E172:

- 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 Sicovit Red 30 E172 at a dose level of 1000 mg/kg bw.

- discolouration of the faeces (red) 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 Sicovit Red 30 E172 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) Sicovit Red 30 E172:

Cmax-levels in plasma of 3.17 μg Fe/g and 4.39 μg Fe/g were noted 0 to 72 hours (tmax as range m/f) after oral administration of 1000 mg Sicovit Red/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.

Applicant's summary and conclusion

Conclusions:

An absolute bioavailability of 0.22%/0.23% (m/f) for Sicovit Red 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.