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Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-11-10 to 2010-02-10
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with limitations which do not impair the overall conclusion from the data
Objective of study:
other: Bioaccessibility
Qualifier:
no guideline followed
Principles of method if other than guideline:
Solubility of test item in simulated human fluids. Principle of test is similar to Transformation/Dissolution testing according to OECD Series 29 (2001)
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2009-06-19
Species:
other: in vitro (simulated human body fluids)
Details on test animals and environmental conditions:
The test media
Five artificial physiological media (phosphate-buffered saline (pH 7.2), Gamble’s solution (pH 7.4), artificial lysosomal fluid (pH 4.5), artificial gastric fluid (pH 1.5) and artificial sweat solution (pH 6.5)), 5 L each one, were were used.
The pH value of each artificial physiological media was measured using a pH meter, previously calibrated with the pH standard solutions.
The saturation in air of each artificial physiological media was measured using a dissolved oxygen meter and corrected for 30 minutes under air flow in order to obtain a value above 70% as dissolved oxygen concentration.
Route of administration:
other: in vitro (simulated human body fluids)
Details on study design:
The dissolved amount of the test item was quantified by the mass concentration of the metals (chromium and iron) in five artificial physiological media (phosphate-buffered saline (pH 7.2), Gamble’s solution (pH 7.4), artificial lysosomal fluid (pH 4.5), artificial gastric fluid (pH 1.5) and artificial sweat solution (pH 6.5)) selected by the Sponsor to simulate relevant human-chemical interactions.
Triplicate samples were prepared for exposure in different test media, each for two different time periods. In addition, one blank sample (without addition of any test item) containing only the test solution was incubated together with triplicate samples for each time period.
Details on dosing and sampling:
Description of the extraction method

Three solutions, 300 mL each one from each test media, were transferred into the 2L plastic containers. The solutions were agitated at 100r.p.m in the mechanical shaker at room temperature for 30minutes. The pH, the temperature and the dissolved oxygen were measured.
In order to measure the blank value two 50 mL blank aliquots were taken from each container, filtered using 0.45µm syringe filters and acidified with 100µL of pure nitric acid before the ICP-MS analysis. These blank samples were stored at 4°C into the refrigerator, until the date of the analysis.

Three 25mg aliquots of the test article were weighted and added to each container solution, labelled A, B and C, preparing a test article solutions with each test media at a solid to liquid ratio of 0.1g/L (example: 25mg in 250mL in order to have a representative weight, using the analytical balance).

Test Media Test vessel Weight [mg]
Phosphate buffered saline (PBS) A 25.4
B 25.1
C 25.0
Artificial sweat solution A 24.9
B 25.4
C 25.6
Artificial interstitial fluid A 25.1
B 25.8
C 25.0
Artificial lysosomal fluid A 25.8
B 25.3
C 25.1
Artificial gastric fluid A 25.1
B 25.9
C 25.3

The solutions were agitated at 100r.p.m in the mechanical shaker at room temperature and at the two sampling times indicated by the Sponsor (2 hours and 24 hours) the pH, the temperature and the dissolved oxygen were measured.

Two 50 mL aliquots from each solution were taken at each sampling time, filtered using 0.45 µm syringe filters, and acidified with 100 µL of pure nitric acid before the ICP-MS analysis. These aliquots were stored at 4°C into the refrigerator, until the date of the analysis.

The dissolved iron and chromium metal ions concentration was measured using the ICP-MS technique.
Toxicokinetic parameters:
other: bioaccessibility
Remarks:
Dissolution of Cr in artifical gastric fluid (pH1.5): 1.2µg/L after 24. Dissolution of Fe 17.5 µg/L (pH1.5) after 24 hours.

52Cr(µg/L)

Artificial gastric fluid

(pH 1.5)

Artificial lysosomal fluid

(pH 4.5)

Artificial sweat solution

(pH 6.5)

Phosphate buffered saline (PBS)

(pH 7.2)

Artificial interstitial fluid

(pH 7.4)

Blank

Mean

 n.d.

2.7

n.d.

n.d.

n.d.

S.D.

-

0.1

-

-

-

CV (%)

-

5.1%

-

-

-

T2h

Mean

<1

n.d.

<1

<1

n.d.

S.D.

-

-

-

-

-

CV (%)

-

-

-

-

-

T24h

Mean

1.2

<1

<1

<1

n.d.

S.D.

0.04

-

-

-

-

CV (%)

3.4%

-

-

-

-

56Fe(µg/L)

Artificial gastric fluid

(pH 1.5)

Artificial lysosomal fluid

(pH 4.5)

Artificial sweat solution

(pH 6.5)

Phosphate buffered saline (PBS)

(pH 7.2)

Artificial interstitial fluid

(pH 7.4)

Blank

Mean

<1

18.4

n.d.

2.0

2.7

S.D.

-

0.8

-

0.1

0.1

CV (%)

-

4.3%

-

5.7%

4.0%

T2h

Mean

7.5

1.4

1.5

<1

<1

S.D.

0.4

0.1

0.3

-

-

CV (%)

4.8%

5.1%

17.7%

-

-

T24h

Mean

17.5

7.9

<1

<1

n.d.

S.D.

0.4

0.4

-

-

-

CV (%)

2.2%

4.5%

-

-

-

n.d.: lower than the method Limit of Detection (L.O.D.) (0.5µg/L for Cr and Fe)

Conclusions:
Interpretation of results (migrated information): other: biologically inert
The dissolution of chromium of the test item Chromium iron oxide is in most cases <1µg/L or below the L.O.D at a loading of 0.1g/L after 2 and 24 hours. Only in artifical gastric fluid (pH1.5) a low concentration of Cr (1.2µg/L) after 24 hours is dissolved.
The dissolution of iron of the test item Chromium iron oxide is in a range of below the L.O.D. (pH 7.4) and 17.5 µg/L (pH1.5) at a loading of 0.1g/L after 2 and 24 hours. A pH dependent dissolution can be observed.
As dissolved Cr and Fe concentrations were below 18 µg/L even at the highest loading of 0.1g/L, referring to a solubility of < 0.018 %, the pigment is considered biologically inert.
Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-07-14 to 2015-07-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Objective of study:
toxicokinetics
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Version / remarks:
2010-07-22
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2014-05-14
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 a tightly closed container
Radiolabelling:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The species was selected for this study design because the rat is a commonly used rodent species for toxicity studies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at administration: males: 54 days; females: 68 days
- Weight at administration: males: 268 - 297 g; females: 227 - 252 g
- Housing: kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm × 23 cm and a height of approx. 18 cm; bedding material: granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany)
- Diet (ad libitum): certified commercial diet (ssniff® R/M-H V1534, ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): drinking water
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- Relative humidity: 55% ± 15% (maximum range).
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
other: chromium iron oxide: oral (gavage); reference item (chromium (III) acetate hydroxide): oral (gavage) and intravenously injected
Vehicle:
other: chromium iron oxide: 0.8 % aqueous hydroxyl propyl methylcellulose gel; reference item (chromium (III) acetate hydroxide): water (oral administration) or 0.9 % NaCl solution (intravenous administration)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
1) Chromium iron oxide
The test item was suspended in the vehicle to the appropriate concentration freshly on the administration day.

2) Reference item (chromium (III) acetate hydroxide)
The reference item was dissolved in the vehicle for oral/intravenous injection.

The administration formulations were continuously agitated by stirring throughout the entire administration procedure.
Administration volume (oral administration / intravenous administration): 10 mL/kg bw

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

The amount of test item and reference item was adjusted to each animal's current body weight on the administration day.
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:
5 males / 5 females
Control animals:
no
Positive control:
none
Details on study design:
- Dose selection rationale: the dose levels for this preliminary study had been selected after consultation with the Sponsor based on available toxicity data:
The oral LD50 value for the reference item was as follows:
chromium acetate, basic: >5000 mg/kg bw
Furthermore, oral bioavailabilities of soluble Cr(III) substances are given in the public domain with approx. 0.1 to 2%.

The test item oral dose of 1000 mg/kg bw corresponds to the limit dose used in a separate 28-day oral toxicity study, which is considered the maximum feasible dose. Based on the chemical composition of the test item, a dose of 1000 mg/kg bw of chromium iron oxide equates to a dose of 212.1 mg Cr/kg bw (corresponding to 820.2 mg Cr3(OH)2(CH3COO)7/kg bw).

The dosage for the reference item administered by intravenous injection was set to 10% of the dose of the reference item adminstered by oral administration on a stoichiometric basis for chromium, thereby lowering the dose for reasons of tolerability of the test animals. This equates to a dose of 21.21 mg Cr/kg bw (corresponding to 82 mg Cr3(OH)2(CH3COO)7/kg bw).

The dose levels for the reference item (oral and intravenous administration) were confirmed in two preliminary experiments each employing two animals per group:

1) First preliminary experiment
Both animals treated once orally with 820.2 mg chromium(III) acetate hydroxide/kg bw revealed slight pilo-erection starting approx. 15 minutes after administration lasting 4 hours.
Both animals treated once intravenously with 82 mg chromium(III) acetate hydroxide/kg bw revealed slight pilo-erection starting approx. 15 minutes after administration lasting 4 hours. Further, haemorrhagic urine was observed within approx. 15 minutes and 1 hour after administration.

2) Second preliminary experiment
Haemorrhagic urine was noted for the two animals treated once with an intravenous injection of 40 mg chromium (III) acetate hydroxide/kg bw at approx. 30 minutes after the administration. The condition of the urine had returned to normal at later checks.
No signs of systemic toxicity were observed for the two animals treated once orally with 400 mg chromium (III) actete hydroxide/kg bw.
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), 1, 2, 4, 8, 12, 24, 48, and 72 hours after administration. The whole blood samples were cooled using an IsoTherm-Rack system until centrifugation. Immediately after centrifugation, the plasma was frozen at -80°C, and stored at this temperature until analysis.
In addition, 4 mL pooled blank plasma (approx. 2 mL per sex) were obtained from spare animals.

A toxicokinetic evaluation of the data on chromium iron oxide plasma levels will be performed. A non-compartment model will be employed. The following parameters will be determined:
AUC0-inf = extrapolated area from zero to infinity
AUC0-t last = extrapolated area from time zero to the last quantifiable plasma concentration >LLOQ
Kel = elimination rate constant
t½ = elimination half-life

Cmax values will be the highest measured plasma concentrations, and tmax values will be the time points of highest plasma concentrations.

Elimination rate constants (Kel) and plasma elimination half-lives (t½) will be 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 will be 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 will be taken to be those at the first blood sampling time.

Furthermore, AUC0-t last-values will be calculated according to the linear trapezoidal rule. Values below or at the lower limit of quantification (LLOQ) will be excluded from the calculation.

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).
- mortality: 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 and on the administration day.

TEST ITEM FORMULATION ANALYSIS
The remaining administration formulations of each test and reference item that was mixed with a vehicle were stored at ≤- 20°C (approximately 5 mL per formulation, in total 3 samples).
Statistics:
The reference item-treated group (oral administration) was compared to the test item treated group.
The following statistical method was used:
STUDENT's t-test: body weight (p ≤ 0.01 and p ≤ 0.05)
The following limits were used:
p = 0.05 / 0.01 about t = 2.3060 / 3.3554
(for 8 degrees of freedom)
Preliminary studies:
Please refer to the field "Details on study design" above.
Toxicokinetic parameters:
other: bioavailability
Remarks:
The relative bioavailability of orally administered pigment was calculated 0.05% (Cr) in relation to a soluble Cr3+compound (Cr3(OH)2(CH3COO)7)injected i.v..
Bioaccessibility testing results:
For Cr a relative bioavailability of 0.94% (mean m/f) was calculated from soluble Cr3(OH)2(CH3COO)7 following oral administration compared to intravenous administration, and of approximately 0.052% (mean m/f) for Cr present in the pigment.

LOCAL TOLERANCE (REFERENCE ITEM; INTRAVENOUS ADMINISTRATION)

None of the animals treated with a single intravenous administration of the reference item revealed any signs of local intolerance reactions at the injection sites as observed during daily visual inspections.

CLINICAL SIGNS, MORTALITY, AND BODY WEIGHT

Chromium iron oxide:

- no abnormalities were observed in behavior or external appearance for any animal treated.

- faeces of all animals were formed normally. A black discolouration was noted for the faeces of 4/5 male animals and 2/5 female animals treated with the test item approx. 8 hours after administration. This is an expected finding being attributed to the high iron content (approx. 47%) of the test item and is not an adverse effect.

- no deaths were noted.

- body weight of all animals was in the expected range on test day 1. The slight difference in the mean body weight between the test item treated group and the reference item groups on test day 1 is a coincidental effect that was due to two test item treated animals with a relatively low body weight gain during the adaptation period.

Reference item (oral administration):

- no abnormalities were observed in behavior or external appearance for any animal treated.

- faeces of all animals were formed normally.

- no deaths were noted.

- body weight of all animals was in the expected range on test day 1.

Reference item (intravenous administration):

- no abnormalities were observed in behavior or external appearance for any animal treated.

- faeces of all animals were formed normally.

- no deaths were noted.

- body weight of all animals was in the expected range on test day 1.

Conclusions:
In a relative bioavailability study, the relative bioavailability of orally administered pigment was calculated 0.05% (Cr) in relation to a soluble Cr3+ compound (Cr3(OH)2(CH3COO)7) injected i.v..
Executive summary:

In conclusion, the oral relative bioavailability of the pigment "Chromium iron oxide" can be assumed to be negligible. The relative bioavailability of orally administered pigment was calculated 0.05% (Cr) in relation to a soluble Cr3+compound (Cr3(OH)2(CH3COO)7)injected i.v..

Endpoint:
basic toxicokinetics, other
Remarks:
mass balance
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-02-06 to 2015-02-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Objective of study:
other: mass balance
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 5 male and 5 female Crl:CD(SD) rats were dosed orally with a singel administration of chromium iron oxide, vehicle (0.8 % aqueous hydroxyl propyl methylcellulose gel), or reference item (chromium (III) acetate hydroxide) via gavage. Clinical signs, mortality and body weight were recorded. Furthermore, urine and faeces of all animals were collected in metabolic cages after administration (sampling period: 0 - 24 hours, 24 - 48 hours, and 48 - 72 hours). Lastly, gross pathology was conducted and organ weights were determined.
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2014-05-14
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 a tightly closed container
Radiolabelling:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The species was selected for this study design because the rat is a commonly used rodent species for toxicity studies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at dosing: males: 49 days; females: 53 days
- Weight at dosing: males: 248.8 - 266.6 g; females: 195.7 - 220.4 g
- Housing (exception: sampling period): kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm × 23 cm and a height of approx. 18 cm; bedding material: granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany)
- Diet (ad libitum): commercial ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): drinking water
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- Relative humidity: 55% ± 15% (maximum range).
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: chromium iron oxide: 0.8 % aqueous hydroxyl propyl methylcellulose gel; reference item (chromium (III) acetate hydroxide): water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
1) Chromium iron oxide
The test item formulation was freshly prepared on the administration day by dissolving the test item in the vehicle to the appropriate concentration. During and after preparation the formulation was stirred and the homogeneity of the suspension was checked by visual appraisal.
Administration volume: 10 mL/kg bw
The administration formulations were continuously agitated by stirring throughout the entire administration procedure.
The amount of the test and reference item was adjusted to the animal's current body weight on the administration day.

2) Reference item (chromium (III) acetate hydroxide; purity: 23.7 %):
The reference item formulation was freshly prepared on the administration day by dissolving the reference item in the vehicle to the appropriate concentration. During and after preparation the formulation was stirred and the homogeneity of the suspension was checked by visual appraisal.
Administration volume: 10 mL/kg bw
The administration formulations were continuously agitated by stirring throughout the entire administration procedure.
The amount of the test and reference item was adjusted to the animal's current body weight on the administration day.
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:
5 males / 5 females
Control animals:
yes, concurrent vehicle
Positive control:
none
Details on study design:
- Dose selection rationale: The dose levels for this study have been selected after consultation with the Sponsor based on available toxicity data:
The oral LD50 value for the reference item was as follows:
chromium acetate, basic: > 5,000 mg/kg bw
Furthermore, oral bioavailabilities of soluble Cr(III) substances are given in the public domain with approx. 0.1 - 2%.

The test item oral dose of 1000 mg/kg bw corresponds to the limit dose used in a separate 28-day oral toxicity study, which is considered the maximum feasible dose. Based on the chemical composition of the test item, a dose of 1000 mg chromium iron oxide/kg bw equates to a dose of 212.1 mg Cr/kg bw (corresponding to 820.2 mg Cr3(OH)2(CH3COO)7/kg bw).

The dose level for the reference item was confirmed in two preliminary experiments employing two animals each (please also refere to IUCLID Section 7.1.1: k_Leuschner_2017_ in vivo). In the first preliminary experiment, both animals were treated once orally with 820.2 mg chromium(III) acetate hydroxide/kg bw and revealed slight pilo-erection starting approx. 15 minutes p.a. lasting for 4 hours. In the second preliminary experiment, both animals were treated once orally with 400 mg chromium(III) acetate hydroxide/kg bw No signs of toxicity were noted.
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine and faeces
- Time and frequency of sampling: all animals of the test item, vehicle and reference item groups were scheduled for urine and faeces sampling. After the single administration, the animals were kept in metabolism cages. Urine and faeces were collected in 3 fractions/animal (sampling periods: 0 - 24 hours, 24 - 48 hours, and 48 - 72 hours).
The urine and faeces weight per collection fraction and animal were determined upon removal of the sample fraction.
All samples were frozen at -20°C or colder and stored at this temperature until analysis.

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).
- mortality: 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 and on the day of administration

GROSS PATHLOLOHY / HISTOPATHOLOGY
- Necrospy and macroscopic inspection: on test day 4 (approx. 72 hours after the administration) the animals were dissected.
The animals were sacrificed, weighed, dissected, and inspected macroscopically.
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland, and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole. The stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intraabdominal lymph nodes, and accessory reproductive organs were recorded.

The weight of the following organs was determined: adrenal gland (2), brain, heart, kidney (2), liver, lungs, lymph nodes (cervical (1), mesenteric (1)), ovary (2), pituitary, prostate, spleen, testicle (2), thymus, and thyroid (1) (including parathyroids).
Paired organs were weighed individually and identified as left or right.

TEST ITEM FORMULATION ANALYSIS
Remaining administration solution (approx. 5 mL) of the test item was stored at ≤- 20°C until analysis (Number of samples: 1).
Statistics:
The test item-treated and reference item-treated groups were compared statistically to the vehicle control group.
The following statistical method was used:
Multiple t-test based on DUNNETT, C. W. New tables for multiple comparisons with a control. Biometrics, 482-491 (Sept 1964): body weight / relative and absolute organ weights (p ≤ 0.01 and p ≤ 0.05)
Preliminary studies:
Please refer to the field "Details on study design" above.
Details on absorption:
Urinary excretion for all elements was negligible and below 0.00009% for Cr, and <0.0024% for Fe. Absorption rate - oral: 0.01 %.
Details on excretion:
Animals that received 1000 mg pigment /kg bw excreted 89.11% Cr and 94.1% Fe of the administered dose via urine and faeces during the first three days after exposure (mean for 10 animals). Within the first 24 hours approximately 85.82% of Cr, and 92.4% of Fe were excreted via faeces as largest fraction. Further 3.04% and 0.25% (Cr), and 0.3% and 1.5% (Fe) were excreted via faeces on the second and third day.
Urinary excretion for all elements was negligible and below 0.00009% for Cr, and <0.0024% for Fe.

CLINICAL SIGNS, MORTALITY, BODY WEIGHT, GROSS PATHOLOGY

Vehicle control group:

- no signs of systemic intolerance was observed

- faeces of control animals were normally formed.

- none of the rats died prematurely.

- individual body weights ranged from 253.4 to 260.3 g for the males and from 195.7 g to 218.2 g for the females on test day 1 and were within the expected range

- no influence was noted on the body weight at autopsy.

- no pathoplogical findings were recorded.

Chromium iron oxide:

- none of the rats treated with the test item showed any changes in behaviour or external appearance.

- faeces of control animals were normally formed.

- none of the rats died prematurely.

- individual body weights ranged from 255.8 to 266.6 g for the males and from 195.7 g to 220.4 g for the females on test day 1 and were within the expected range

- no influence was noted on the body weight at autopsy.

- no test item-related changes were noted for the animals treated with the test item at macroscopic inspection at necropsy.

- no test item-related changes in relative and absolute organ weights were noted for the animals treated with the test item.

- statistically significant differences in organ weights compared to vehicle control group which are not considered to be test item-related are as follows:

females (test day 4): increased relative thymus weight and increased absolute thymus weight (p ≤ 0.01)

males (test day 4): increased absolute left testis weight, increased absolute heart weight, and increased absolute pituitary weight (p ≤ 0.05)

 

Reference item:

- none of the rats treated with the reference item showed any changes in behaviour or external appearance.

- faeces of control animals were normally formed.

- none of the rats died prematurely.

- individual body weights ranged from 248.8 to 258.2 g for the males and from 196.7 g to 216.1 g for the females on test day 1 and were within the expected range

- no influence was noted on the body weight at autopsy.

- no reference item-related changes were noted for the animals treated with the reference item at macroscopic inspection at necropsy. An enlarged left thyroid was noted for one female animals (not considered reference item-related).

- no reference item-related changes in relative and absolute organ weights were noted for the animals treated with the reference item.

- statistically significant differences in organ weights compared to vehicle control group which are not considered to be reference item-related are as follows:

males (test day 4): increased relative thyroid / parathyroid weight and increased absolute thyroid / parathyroid weight (p ≤ 0.05)

Conclusions:
The mass balances for Cr and Fe are essentially complete and indicate that the elements contained in the pigment "Chromium iron oxide", present as Cr3+and Fe3+, are not absorbed in the gastrointestinal tracts to any significant extent, but pass the animal effectively unchanged. 
Executive summary:

Animals that received 1000 mg pigment /kg bw excreted 89.11% Cr and 94.1% Fe of the administered dose via urine and faeces during the first three days after exposure (mean for 10 animals). Within the first 24 hours approximately 85.82% of Cr, and 92.4% of Fe were excreted via faeces as largest fraction. Further 3.04% and 0.25% (Cr), and 0.3% and 1.5% (Fe) were excreted via faeces on the second and third day.

Urinary excretion for all elements was negligible and below 0.00009% for Cr, and <0.0024% for Fe.

The mass balances for Cr and Fe are essentially complete and indicate that the elements contained in the pigment "Chromium iron oxide", present as Cr3+and Fe3+, are not absorbed in the gastrointestinal tracts to any significant extent, but pass the animal effectively unchanged. 

Description of key information

The in-vitro and in-vivo experiments described above are in very good agreement with regards to the negligible level of bioavailability of the elements Cr and Fe contained in the pigment.

(1)   In in-vitro dissolution experiments in five different artificial physiological media, dissolved Cr and Fe concentrations were below 18 µg/L even at the highest loading of 0.1g/L, corresponding to a solubility of 0.018 %.

(2)   In a 28-day oral toxicity study with 1,000 mg/kg pigment no increase in Cr and Fe plasma and urine concentrations were observed when sampled at the end of the 28-day exposure period. From a final dose of 1,000 mg/kg of the pigment that the animals received on the last day of the study, only cumulated relative amounts of < 0.0025 % (m/f) were found in the terminal 24-h urine collection period.

(3)   In a mass balance study with a single oral dose of 1,000 mg/kg of the pigment, 89.1% Cr, and 94.1% Fe of the dose were excreted via faeces within 3 days, with only <0.0024% of the dose being excreted via urine at the same time.

(4)   In a relative bioavailability study, the relative bioavailability of orally administered pigment was calculated 0.05% (Cr) in relation to a soluble Cr3+compound (Cr3(OH)2(CH3COO)7)injected i.v..

Comparing the findings ofin-vitrodissolution testing (1) within-vivoresults (2-4), thein-vivodata consistently demonstrates slightly lower bioavailability. This is in agreement with the general understanding thatin-vitroexperiments in simulated gastric juice provide a conservative estimate of actual (in-vivo) bioavailability.

In conclusion, the oral relative bioavailability of the pigment "Chromium iron oxide" can be assumed to be negligible, as demonstrated in three independent in-vivo studies in rats yielding very comparably results supported by anin-vitrodissolution experiment in five different artificial physiological media.

A rounded value of <0.01% for oral absorption can be taken forward from (i) terminal urine/plasma sampling in a study involving 28 repeated oral doses of 1,000 mg pigment/kg bw/d (<<0.008% for both metals) and (ii) a mass balance study involving a single dose of 1,000 mg pigment/kg bw (0.00009% for Cr, and <0.0024% for Fe).

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

The toxicity data in this registration dossier refer explicitly to the pigment Chromium iron oxide and document its negligible bioavailability and the complete lack of any human health hazard. Experiments on the bioavailability of the pigment are summarised and discussed in this section.

 

Summary ofin-vitrobioaccessibility experiments (Pardo Martinez, 2010)

The chemical and physiological properties of the pigment chromium iron oxide are 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 mexposure 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.

 The dissolution of chromium of the test item chromium iron oxide is in most cases 1µg/L or below the L.O.D at a loading of 0.1g/L after 2 and 24 hours. Only in artifical gastric fluid (pH 1.5) a low concentration of 1.2µg/L after 24 hours is dissolved.

The dissolution of iron of the test item chromium iron oxide is in a range of below the L.O.D. (pH 7.4) and 17.5 µg/L (pH 1.5) at a loading of 0.1g/L after 2 and 24 hours. A pH dependent dissolution can be observed. In conclusion, since the dissolved Cr and Fe concentrations were below 18 µg/L even at the highest loading of 0.1g/L, referring to a solubility of 0.018 %, this pigment may reasonably be considered biologically inert.

 

Toxicokinetic screening data from a 28 day repeated dose oral toxicity study (Leuschner, 2017)

In a 28 day repeated dose toxicity study, male and female rats were given a daily dose of the pigment "Chromium iron oxide" of 1,000 mg/kg bw/day via gavage. Individual urine samples were collected from all animals prior to sacrifice in one cumulated 24-h fraction/animal after the last oral application, and blood samples were collected from each animal upon sacrifice. The plasma and urine samples were analysed for total chromium and iron content.

The uptake of chromium and iron during a 24 hour urine and plasma sampling period was demonstrated to be negligible considering that <<0.00005 of the dose was excreted via urine for both metals, mirrored by either minimal or no increases in blood plasma concentrations.

The chromium and iron concentrations of the 24h-urine samples, collected during the day before final sacrifice, ranged from: 5.87 - 134.17μg/L urine(mean: 47.2 ± 53.2) and 3.16 - 18.3μg/L urine(mean: 10.3 ± 7.30) for Cr and from 36.9 - 77.6μg/L urine (mean: 49.5 ± 16.4) and 33.3 - 70.6μg/L urine (mean: 45.6 ± 15.3)for Feforthe male and female animals of the control group, respectively.

For the dosed group, the concentrations were2.38 - 823μg/L urine (mean: 169 ± 366) and 2.66 - 13.1μg/L urine (mean: 5.47 ± 4.44) for Cr and 25.6 - 88.1μg/L urine (mean: 45.0 ± 25.1) and 31.1 - 61.0μg/L urine (mean: 47.1 ± 11.0) for Fefor the male and female animals, respectively.

Following a subtraction of the background urinary element excretion (control group), and taking into account the excreted urine volume (mean 12.1 mL (m) and 8.5 mL (f)) and the body weight of the animals at the end of the study (mean 338.9 g (m) and 223.1 g (f)), the following conclusion can be made:

From a final dose of 1,000 mg/kg of the pigment that the animals received on the last day of the study, only cumulated relative amounts of 0.0022 % (m) or 0.00005% (f) were found in the terminal 24-h urine collection period.

 

Summary of comparative Mass-Balance Study (Leuschner 2017a):

In a comparative mass balance study involving oral dosing of (i) the inorganic pigment "Chromium iron oxide" and (ii) a soluble salt of the element contained therein (Cr3+), the gastrointestinal absorption as well as urinary and faecal excretion were compared, plus consideration of dietary „background“ intake/excretion via a vehicle-dosed control. For details, please refer to the corresponding robust study summary.

In brief, 10 (5m/5f) animals per group received a single oral dose of 1000 mg/kg of the pigment or820.2 mgCr3(OH)2(CH3COO)7/kg bw(corresponding to 212.1 mg/kg Cr). A third group served as vehicle treated control. Animals were individually housed in metabolic cages and daily samples of urine and faeces were collected for three days. All samples were analysed for chromium. The averaged “background” excretion via urine and faeces of the control animals was subtracted from the amounts excreted by the dosed animals, and a mass balance was calculated.

Animals that received a 212.1 mg Cr/Kg bw (administered as Cr3(OH)2(CH3COO)7excreted 83 % (Cr), of the administered dose (as mean, male and female animals) via urine and faeces during the first three days after exposure.

The largest fraction (78.92 % for Cr) was excreted via faeces and urine (0.35% for Cr) already within the first 24h.

Approximately 17 % of the administered dose for Cr remain unaccounted for in this group. Due to the fact that Cr3+has a very low absorbance ability within the g.i. tract (~0.1-2%) a hypothesis that this fraction was contained in the exsanguinated blood of the animals or has distributed to the organs seems not appropriate. It may be considered that delayed excretion of the whole dose is reasonable since at 72h still ~0.32% of the Cr dose (mean, mainly female animals) was excreted.

Further it may be considered that the actually received dose did not fully correspond to the nominal dose as calculated. These aspects were not further addressed within the context of this study.

Animals that received 1000 mg pigment /kg bw excreted 89.11% Cr and 94.1% Fe of the administered dose via urine and faeces during the first three days after exposure (mean for 10 animals). Within the first 24 hours approximately 85.82% of Cr, and 92.4% of Fe were excreted via faeces as largest fraction. Further 3.04% and 0.25% (Cr), and 0.3% and 1.5% (Fe) were excreted via faeces on the second and third day.

Urinary excretion for all elements was negligible and below 0.00009% for Cr, and <0.0024% for Fe.

In total, the mass balances for Cr and Fe are essentially complete and indicate that the elements contained in the pigment "Chromium iron oxide", present as Cr3+and Fe3+, are not absorbed in the gastrointestinal tracts to any significant extent, but pass the animal effectively unchanged. 

 

Summary of relative bioavailability study (Leuschner 2017b):

A relative bioavailability study involving serum kinetics over a period of 72 hours p. a. involving an i. v. dosing of a soluble Cr reference substance (Chromium(III) acetate hydroxide) compared to single oral doses of the same substance and the pigment was performed. For details, please refer to the corresponding robust study summary. In brief, 10 animals (5m/5f) per group received single doses of (1) 0.83 mg/kg Cr3(OH)2(CH3COO)7intravenously, (2) of 820.2 mg/kg Cr3(OH)2(CH3COO)7via oral gavage, and (3) 1000 mg/kg of the pigment via oral gavage.

Blood samples were taken at 0, 1, 2, 4, 8, 12, 24, 48 and 72 hours post exposure and blood plasma samples were prepared and analysed for the element Cr.

Cmax-levels in plasma of 0.16 µg Cr/g and 0.55 µg Cr/g were noted ~15.8 or ~2.8 hours (m/f) after intravenous administration of 0.83 mg/kg Cr3(OH)2(CH3COO)7for the male and female rats on test day 1, respectively.

Furthermore, Cmax-levels of 1.01 µg Cr/g and 1.20 µg Cr/g were noted ~2, or 2.2 hours (m/f) after oral administration of 820.1 mg/kg Cr3(OH)2(CH3COO)7for the male and female rats on test day 1, respectively.

Lastly, Cmax-levels of 0.002 µg Cr/g and 0.212 µg Cr/g were noted ~4 and 16 hours (m/f) after oral administration of 1000 mg pigment/kg for the male and female rats on test day 1, respectively. For comparison, the average (n=30) concentration of Cr in plasma taken before exposure at t=0 h was 0.142 µg Cr/g plasma.

Due to the very low Cr levels determined after blank subtraction, no elimination half-life could be calculated. The plasma concentrations remained more or less constant in range of the background levels during the whole measurement period. 

For Cr a relative bioavailability of 0.94% (mean m/f) was calculated from soluble Cr3(OH)2(CH3COO)7following oral administration compared to intravenous administration, and of approximately 0.052% (mean m/f) for Cr present in the pigment.

In sum, experimentally determined plasma kinetic values for Cr are very consistent and the very low relative bioavailability of Cr from the pigment demonstrates that the pigment can be considered inert without any systemic hazard potential for human health.

Overall conclusion:

The in-vitro and in-vivo experiments described above are in very good agreement with regards to the negligible level of bioavailability of the elements Cr and Fe contained in the pigment.

(1)   In in-vitro dissolution experiments in five different artificial physiological media, dissolved Cr and Fe concentrations were below 18 µg/L even at the highest loading of 0.1g/L, corresponding to a solubility of 0.018 %.

(2)   In a 28-day oral toxicity study with 1,000 mg/kg pigment no increase in Cr and Fe plasma and urine concentrations were observed when sampled at the end of the 28-day exposure period. From a final dose of 1,000 mg/kg of the pigment that the animals received on the last day of the study, only cumulated relative amounts of < 0.0025 % (m/f) were found in the terminal 24-h urine collection period.

(3)   In a mass balance study with a single oral dose of 1,000 mg/kg of the pigment, 89.1% Cr, and 94.1% Fe of the dose were excreted via faeces within 3 days, with only <0.0024% of the dose being excreted via urine at the same time.

(4)   In a relative bioavailability study, the relative bioavailability of orally administered pigment was calculated 0.05% (Cr) in relation to a soluble Cr3+compound (Cr3(OH)2(CH3COO)7)injected i.v..

Comparing the findings ofin-vitrodissolution testing (1) within-vivoresults (2-4), thein-vivodata consistently demonstrates slightly lower bioavailability. This is in agreement with the general understanding thatin-vitroexperiments in simulated gastric juice provide a conservative estimate of actual (in-vivo) bioavailability.

In conclusion, the oral relative bioavailability of the pigment "Chromium iron oxide" can be assumed to be negligible, as demonstrated in three independent in-vivo studies in rats yielding very comparably results supported by anin-vitrodissolution experiment in five different artificial physiological media.

A rounded value of <0.01% for oral absorption can be taken forward from (i) terminal urine/plasma sampling in a study involving 28 repeated oral doses of 1,000 mg pigment/kg bw/d (<<0.008% for both metals) and (ii) a mass balance study involving a single dose of 1,000 mg pigment/kg bw (0.00009% for Cr, and <0.0024% for Fe).

Absorption rate - oral: 0.01 %