Zirconium iron pink zircon

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-01-16 to 2015-02-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008-10-03

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2014-05-14

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Chromium iron oxide

EC Number:

235-790-8

EC Name:

Chromium iron oxide

Cas Number:

12737-27-8

Molecular formula:

Fe(x)Cr(2-x)O3 0,65≤x≤1,75

IUPAC Name:

Chromium iron hematite

Test material form:

solid: particulate/powder

Details on test material:

- Chemical description: Chromium iron oxide
- Substance type: inorganic pigment
- State of aggregation: solid, black powder, odourless, Hematite-corundum structure
- Storage condition of test material: at room temperature

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

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

Rats were selected because of their proven suitability in toxicology studies and to comply with regulatory requirements for testing in a rodent animal species.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at first dosing: males: 35 days; females: 36 days
- Weight at first dosing: males: 145.3 g - 164.2 g; females: 134.2 g - 149.8 g
- Housing: kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 x 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 V1530 diet (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): tap water
- Acclimation period: 9 days

DETAILS OF FOOD AND WATER QUALITY: no contaminants above the limitiations were noted for drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C ± 3 °C (maximum range)
- Relative humidity: 55 % ± 15 % (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The route of administration was selected according to the expected route of exposure.

Vehicle:

other: 0.8 % aqueous hydroxyl propyl methylcellulose gel

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in the vehicle to the appropriate concentration. The administration formulation was continuously agitated by stirring throughout the entire administration procedure.
The administration formulation was freshly prepared every day.
Administration volume: 10 mL/kg bw/day
The amount of the test item was adjusted to each animal's current body weight daily.

VEHICLE
- Source: FAGRON GmbH & Co. KG, 22885 Barsbüttel, Germany
- Batch no.: 12G23-N03

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the test item that was mixed with the vehicle, tests by ICP-OES were conducted to determine the concentration, stability and homogeneity of the test item in the formulations (Fraunhofer IME, report no. EBR-151/6-27/y).
For the analysis of the test item-vehicle mixtures, samples of approximately 10 mL were taken at the following times and stored at ≤-20 °C:

1) At study initiation:
- analysis of stability and concentration: immediately after preparation of the administration formulation as well as after 8 and 24 hours storage at room temperature (number of samples: 3).
- homogeneity: at the start of administration, during (middle) administration and before administration to the last animal of the test item treated group (number of samples: 3).

2) at study termination:
- analysis of concentration: during treatment always before administration to the last animal of the test item treated group (number of samples: 1).

For the test item a digestion method was developed before (for faeces samples in study
EBR-151/6-27). In case of this pigment a nitric acid microwave assisted digestion with a higher temperature was most efficient for small amounts of the pigment. In the first part of the test a small amount 0.1 mL was used for the digestion but the results of this procedure showed a low recovery of the nominal amount of pigment (according to LPT 100 g pigment / L). A possible explanation for this low recovery could be the difficulty to take off only 100 µL of the test item solution due to the viscosity of the solution which results in a possible inhomogeneity in the taken solution. Due to this fact a different procedure was chosen for the digestion.
In the second step a real “total digestion” was performed. For this the total remaining material was used in a sequential digestion. A detailed description of the procedure is given in the section digestion. Just in short the total solution was transferred in a new vial and concentrated nitric acid was added to the solution. Afterwards the solution was shaken and centrifuged. After centrifugation the supernatant was removed and the remaining pigment was given in three digestion vials. Afterwards concentrated nitric acid was added and a modified microwave digestion was performed. After digestion the supernatant was removed with a pipette and to the remaining pigment new concentrated nitric acid was added. These steps were performed until only less to mainly no pigment was visible. Due to the fact that the pigment Chromium iron oxide shows a low digestion rate during the microwave digestions a HNO3/HF mixture step was inserted after 18 former HNO3 digestion to investigate if a HNO3/HF step would be more effective in the end. After this digestion step still a high amount of pigment was visible. For this reason the more toxic HNO3/HF digestion was not performed further and the “normal” HNO3 digestion was used until no pigment was visible any more.

Samples of digested application solutions (test item-vehicle mixtures) were measured by ICP-OES. The ICP-OES measurements were performed with an Agilent 720 ICP-OES (Agilent Technologies, Waldbronn, Germany). Chromium was detected at the wavelength 206.158 nm, 267.716 nm, 276.653 nm and 357.868 nm; iron was detected at the wavelength 238.204 nm, 241.052 nm, 259.837 nm and 259.940 nm. The following solutions were used to calibrate the instrument: blank, 1 µg/L, 2.5 µg/L, 5 µg/L, 7,5 µg/L, 10 µg/L, 25 µg/L, 50 µg/L, 75 µg/L, 100 µg/L, 250 µg/L, 500 µg/L, 750 µg/L and 1000 µg/L. Calibrations were performed before each measurement. The calibration formula was calculated using the linear regression algorithm of the ICP-OES instrument. The respective wavelength data with the best recoveries for the validation samples (certified reference material, quality control standards, recalibration standards and fortifications) in the measurement series and a correlation coefficient with at least 0.995 were used for calculating concentrations. Correlation coefficients (r) for the wavelengths used for evaluation of data were at least 0.997100. For each sample, at least three internal measurements were performed and the mean was calculated and printed by the instrument software. Samples were diluted for adaption to the calibration matrix and to fit into the calibration curve.

Instrumental and analytical set-up for the ICP-OES instrument:
Agilent 720, Agilent Technologies, Waldbronn, Germany
Nebulizer: Sea spray nebulizer from Agilent
Spray chamber: Glass cyclonic spray chamber from Agilent
Carrier gas flow: 0.75 L/min
RF power: 1200W
Wavelengths:
Cr: 206.158 nm, 267.716 nm, 276.653 nm and 357.868 nm
Fe: 238.204 nm, 241.052 nm, 259.837 nm and 259.940 nm

The applied LOD/LOQ calculations for the Agilent 720 ICP-OES are (according to DIN 32645) [6]:
LOD: 3 * standard deviation of calibration blank/slope of the calibration
LOQ: 3 * LOD

The resulting LODs/LOQs are as follows:
- LOD: 0.044-1.20 µg/L (Fe); 0.09-0.905 µg/L (Cr)
- LOQ: 0.133-3.59 µg/L (Fe); 0.271-2.71 µg/L (Cr)
- correlation coefficient: 0.999986 (Fe); 0.999989 (Cr)
The certified reference material TMDA-52.4 and TMDA-54.5 as well as quality control standards and recalibration standards were analyzed as quality assurance samples along with the test samples. To meet quality assurance requirements recovery needs to be in the range of ± 15 % of the respective certified value.
Selected samples were fortified with a known amount of chromium and iron (by standard addition of commercial standards) to determine the standard recovery of chromium and iron. For fortified samples, recoveries were 96.3 - 102% for Cr and 91.2 - 102% for Fe.

Results:
Dose verification:
nominal dose: 1,000 mg/kg bw pigment (212 mg/kg bw Cr, 466 mg/kg bw Fe)
Results:
Analysis of stability and concentration (3 samples):
Recovery [%]:
Cr: 73.0 - 83.9
Fe: 76.6 - 88.3

Anaylsis of homogenity (3samples):
Recovery [%]:
Cr:77.9 - 83.8
Fe: 82.9 - 89.2

Anaylsis of concentration (1sample):
Recovery [%]:
Cr: 105.8
Fe: 111.1

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Doses / concentrations

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

Details on study design:

- Dose selection rationale: in agreement with the Sponsor and based on available toxicity data a limit test was performed.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule (clinical signs): before and after dosing at each time of dosing as well as regularly throughout the working day from 7.30 a.m. to 4.30 p.m. and on Saturdays and Sundays from 8.00 a.m. to 12.00 noon with a final check performed at approx. 4.00 p.m.
- Time schedule (mortality): early in the morning and again in the afternoon of each working day as well as on Saturdays and Sundays with a final check at approx 4.00 p.m.
- Cage side observations checked: clinical signs & mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure and once a week thereafter (1, 2, 4, 8 and 24 hours after administration) as well as in test week 4 prior to any laboratory investigations.

BODY WEIGHT: Yes
- Time schedule for examinations: at the time of group allocation, on the day of commencement of treatment and once a week thereafter (always on the same day of the week)

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment week.
The relative food consumption (in g/kg bw/day) was determined
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the start of administration and at the end of test week 4
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at study termination (on the day of dissection)
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters examined: haemoglobin content, erythrocytes, leucocytes, differential blood count (relative and absolute; neutrophilic granulocytes, eosinophilic granulocytes, basophilic granulocytes, lymphocytes, monocytes, and large unstained cells), reticulocytes, platelets, haematocrit value, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, thromboplastin time, and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at study termination (on the day of dissection)
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters examined: albumin, globulin, albumin/globulin ratio, bile acids, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), urea (blood), calcium, chloride, potassium, sodium, alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, and lactate dehydrogenase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in test week 4 approx. 1 to 2 hours after dosing and before any blood sampling
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory reactivity / grip strength / motor activity
1) Observational screening: righting reflex, body temperature, salivation, startle response, respiration, mouth breathing, urination, convulsions, pilo-erection, diarrhoea, pupil size, pupil response, lacrimation, impaired gait, stereotype, toe pinch, tail pinch, wire maneuver, hind leg splay, positional passivity, tremors, positive geotropism, limb rotation, and auditory function
2) Functional tests: grip strength and locomotor activity

IMMUNOLOGY: No

TOXICOKINETC: Yes (please refer to Fraunhofer IME, report no. EBR-151/6-27/y)
Urine and plasma samples were obtained at study termination. Urine and plasma samples were analysed for chromium and iron levels by ICP-OES and ICP-MS.
- urine sample: individual urine samples were collected from all animals before scheduled sacrifice following the last administration on test day 28. The animals were placed in metabolic cages during a 24-hour collection period, directly after the last oral administration. The urine weight/animal was determined upon removal of the sample. Pooled blank urine were obtained from spare animals.
- plasma sample: on the scheduled day of sacrifice, a terminal blood sample was collected from all animals under isoflurane anaesthesia in order to obtain LiHeparin plasma/animal. Afterwards, the animals were sacrificed and dissected.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

On test day 29 (approx. one day after the last administration), the animals were sacrifice and macroscopically inspected. All superficial tissues were examined visually and by palpation and the cranial roof was 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 and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

The weights of the following organs of all animals were determined before fixation: adrenal gland (2), brain, epididymis (2), heart, kidney (2), liver, ovary (2), spleen, testicle (2), thymus, as well as prostate and seminal vesicles with coagulating glands as a whole.
Paired organs were weighed individually and identified as left or right.

The following organs or parts of organs of all animals were fixed in 7% buffered formalin (exceptions: eyes fixed in Davidson's solution and testes in Bouin's solution): adrenal gland (2), bone (os femoris with joint), bone marrow (os femoris), brain (3 levels: cerebrum, cerebellum, medulla/pons), epididymis (2), eye with optic nerve (2), gross lesions observed, heart (3 levels: right and left ventricle, septum), large intestine (colon, rectum), small intestine (duodenum, jejunum, ileum, incl. Peyer´s patches; Swiss roll method), kidney and ureter (2), liver, lungs (with mainstem bronchi and bronchioles (preserved by inflation with fixative and then immersion)), lymph node (1, cervical), lymph node (1, mesenteric), mammary gland (male and female), muscle (skeletal, leg), nerve (sciatic), ovary (2), pituitary, prostate and seminal vesicles with coagulating glands, spinal cord (3 sections), spleen, stomach, testicle (2), thymus, thyroid (2) (incl. parathyroids), tissue masses or tumours (incl. regional lymph nodes), trachea (incl. larynx), urinary bladder, uterus (incl. cervix and oviducts), and vagina

The above-listed organs of all animals were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining.
In addition, frozen sections of the heart, liver and one kidney were made, stained with Oil Red O and examined microscopically.
Parathyroids cannot always be identified macroscopically. They were examined microscopically if in the plane of section and in all cases where they were noted as grossly enlarged.

Statistics:

The test item-treated group was compared with the vehicle control group:
The following statistical methods were used:

1) STUDENT's t-test: all numerical functional tests / body weight / food consumption / haematology and coagulation / clinical biochemistry / relative and absolute organ weights (p ≤ 0.05 and p ≤ 0.01)
The following limits were used:
p = 0.05/0.01 about t = 2.3060/3.3554 (for 8 degrees of freedom)

2) Exact test of R. A. FISHER: histology (p ≤ 0.05 and p ≤ 0.01)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

CLINICAL SIGNS
- no changes in behaviour or external appearance were noted for the male and female rats treated with 1000 mg chromium iron oxide/kg bw/day or for the animals treated with the vehicle control.
- all male and female rats treated with 1000 mg Chromium iron oxide/kg bw/day revealed black discoloured faeces as of test day 8 (not an adverse effect; finding is considered to be due to the test item (black powder)).
- faeces of the control and test item-treated animals were formed normally.

MORTALITY
- none of the animals died prematurely during the study.

BODY WEIGHT AND WEIGHT CHANGES
- no test item-related influence was observed for the body weight, the body weight gain and body weight at autopsy in the male and female rats treated with 1000 mg chromium iron oxide/kg bw/day (all data are regarded to be within the normal range).

FOOD CONSUMPTION AND COMPOUND INTAKE
- no test item-related changes in relative food consumption were noted for the male and female rats treated with 1000 mg chromium iron oxide/kg bw/day compared to the control group.

WATER CONSUMPTION AND COMPOUND INTAKE
- visual appraisal of the drinking water consumption did not reveal any test item-related influence.

OPHTHALMOLOGICAL FINDINGS
- ophthalmological examination revealed no changes of the eyes and the optic region in the male and female rats treated with 1000 mg chromium iron oxide/kg bw/day or for the animals treated with the vehicle control.

HAEMATOLOGICAL FINDINGS
- no test item-related influence in haematological and coagulation parameters was noted for the male and female rats treated with 1000 mg chromium iron oxide/kg bw/day compared to the control group.
- statistically significant differences (p ≤ 0.05) in a haematological parameters of test item-treated animals compared to the control animals was recorded (no test item-related findings):
females (test day 29): decreased haemoglobin content (control group: 9.84 ± 0.27 mmol/L vs. treatment group: 9.42 ± 0.26 mmol/L) and increased absolute basophilic granulocytes (control group: 0.010 ± 0.000 x10³/µL vs. treatment group: 0.016 ± 0.005 x10³/µL)
However, the stated haematological findings are within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below). These findings should therefore not be regarded as adverse response but as normal biological variation.

CLINICAL BIOCHEMISTRY FINDINGS
- no test item-related influence in biochemical parameters was noted for the male and female rats treated with 1000 mg Chromium iron oxide/kg b.w./day once daily for 28 days compared to the control group.
- statistically significant differences (p ≤ 0.05) in a biochemical parameters of test item-treated animals compared to the control animals was recorded (no test item-related findings):
males (test day 29): increased cholesterol (control group: 1.084 ± 0.142 mmol/L vs. treatment group: 1.564 ± 0.442 mmol/L) and increased potassium (control group: 3.582 ± 0.151 mmol/L vs. treatment group: 3.826 ± 0.078 mmol/L)
females (test day 29): decreased sodium (control group: 139.6 ± 0.5 mmol/L vs. treatment group: 138.2 ± 0.8 mmol/L)
However, the stated biochemical findings are within in the normal range, typical for that strain and the age of the animals (see attached historical control data of the lab in the field "Attached background material" below). These findings should therefore not be regarded as adverse response but as normal biological variation.

BEHAVIOUR (FUNCTIONAL FINDINGS)
- neurological screening did not reveal any test item-related influence in the male and female rats treated with 1000 mg test item/kg bw/day
- examination results of the animals treated with the vehicle control were also in the normal range.
- statistically significant difference (p ≤ 0.05) in a neurological parameter of a test item-treated animal compared to the control animals was recorded (no test item-related finding):
males (test week 4): increased forelimb grip strength
The mean grip strength of the forelimb of both control (371.5 ± 60.5 g) and treated (499.3 ± 63.3 g) animals is at the upper limit or outside the mean historical control range (85.3 - 395.0 g). This might be a cause of the highly fluctuating values within the individual values, even within the individual values for the identical animal (range of forelimb grip strength for control: 159-714 g and treated: 176-750 g animals). Based on this it is concluded that within the historical control and considering the high fluctuation of the grip-strength values, the statistical significance need to be regarded as chance finding without any biological significance.
Individual data and historical control data for forelimb grip strength can be found in the field "Attached background material" below).

ORGAN WEIGHT FINDINGS INCLUDING ORGAN / BODY WEIGHT RATIOS
- no test item-related changes in relative and absolute organ weights were noted for the male and female rats treated with 1000 mg chromium iron oxide/kg bw/day compared to the control group.
- statistically significant differences in organ weights of test item-treated animals compared to the control animals was recorded (no test item-related finding): males (test day 29; p ≤ 0.01 and p ≤ 0.05): increased absolute brain weight (control group: 1.860 ± 0.067 g vs. treatment group: 2.000 ± 0.047 g), increased absolute kidney weight (left)(control group: 1.110 ± 0.099 g vs. treatment group: 1.366 ± 0.206 g), increased absolute kidney weight (right)(control group: 1.186 ± 0.120 g vs. treatment group: 1.388 ± 0.145 g), and increased absolute liver weight (control group: 8.00 ± 0.74 g vs. treatment group: 9.52 ± 1.25 g)
However, the relative organ weight of the respective organ of the males was unaffected and the reported values for the absolute organ weights are within the normal range for that rat strain and age of the animals. These findings should therefore not be regarded as adverse response but as normal biological variation (see attached historical control data of the lab in the field "Attached background material" below).
females (test day 29; p ≤ 0.05): increased relative ovary weight (right)(control group: 0.1837 ± 0.0447 g vs. treatment group: 0.2590 ± 0.0380 g) and increased absolute ovary weight (right)(control group: 0.0374 ± 0.0104 g vs. treatment group: 0.0516 ± 0.0084 g)

GROSS PATHOLOGICAL FINDINGS
- none of the male and female rats treated with 1000 mg test item/kg bw/day revealed any test item-related macroscopic changes at necropsy on test day 29.
- 2/5 male and 3/5 female animals treated with 1000 mg chromium iron oxide/kg bw/day revealed a green discoloured content of the intestines (caecum, colon and rectum)(not an adverse effect; finding is considered to be due to the test item).

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC
- histomorphological examination did not reveal any morphological changes which are considered to be related to the administration of the test item (no difference between the groups).
- granular black material in the mucus in the intestine-lumen of the male and female rats of test item treated group appeared to be test substance. It was not observed in the control animals. This material did not cause any damages to the intestine-epithelium. This finding correlated with the macroscopic findings.
- inflammatory lesions in different organs are considered to be coincidental findings or spontaneous organ changes and are thus not test item-related (no differences were noted between the groups).
- fatty infiltration in the hepatocytes and in the tubular epithelial cells of the kidneys in male and female rats of the control and test item-treated groups were within the physiological limits.
- involution of the thymus in the rats of both groups corresponded in type, incidence and severity to the age of the animals.
- coincidental findings from different organs in a small number of control and test item-treated animals are considered to be spontaneous organ changes and are thus not test item-related.

TOXICOKINETICS
Chromium and iron are of negligible bioavailability from the test substance Chromium iron oxide: by recalculating the urine levels and setting them into relation to the administered dose of the individual elements Cr and Fe, it is reasonable to assume that the majority of the dose (>99.9%) represents non-absorbable, “inert” pigment, likely to be excreted via faeces. Please also refer to the field "Attached background material" below.
Furthermore, there were either no appreciable or only negligible increases in blood plasma levels for both metals.

Effect levels

Key result

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

NOAEL (oral; rats) > 1000 mg chromium iron oxide/kg bw/day

No test item-related changes were observed for clinical signs, mortality, neurologically screening, body weight/body weight gain, food consumption, water consumption, haematology, clinical chemistry, organ weights, ophthalmology, gross pathology, and histopathology.
The uptake of chromium and iron during a 24 hour urine and plasma sampling period was demonstrated to be negligible considering that <<0.003% of the dose was excreted via urine for all two metals, mirrored by either minimal or no increases in blood plasma concentrations. This supports the assumption that two elements are not biologically available upon ingestion of the pigment Chromium iron oxide.