Registration Dossier

Administrative data

Description of key information

It is concluded that the pigment was well tolerated and that no signs of systemic toxicity whatsoever were observed in rats when administered at a dose of 1000 mg/kg bw/day for up to 28 days. Either no or only marginal increases in Cr and Fe plasma concentrations were observed, and only a minor fraction (<0.002%) of the total administered dose of Cr and Fe was collected via urine, documenting the lack of bioavailability of this pigment. The no observed adverse effect level (NOAEL) in rats is 1000 mg/kg/day.

 

In a 90 -day repeated dose toxicity by inhalation study, rats were exposed via nose-only inhalation towards aerosol concentrations of 0.6, 2.5 and 10 mg/m³ of chromium iron oxide. All animals survived the test period and were euthanized at scheduled dates. Effects indicating local or systemic toxicity were not observed. Sex-specific differences were not detected. The NOAEC was considered to be above the highest concentration of 10 mg/m³ (the concentration at or above the onset of lung overload, thus constituting the maximum tolerated concentration).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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
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
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
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
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
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
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)
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.
Key result
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
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.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
start: 2020-07-30 and still ongoing
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study
Version / remarks:
2018-06-25
Deviations:
yes
Remarks:
Ophthalmology not performed (this endpoint is not sensitive in particle studies); urine analysis not performed (endpoint optional only in guideline)
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP certificate signed 2018-11-22
Limit test:
no
Species:
rat
Strain:
other: Crl:WI (Han)
Details on species / strain selection:
Wistar rats are commonly used in subchronic and chronic inhalation toxicity studies. They fulfil the criteria stated by a U.S. EPA Workshop (Vu et al., 1996)* such as (i) a low background rate of neoplasia, (ii) a low background rate of pulmonary disease, (iii) longevity, and (iv) a history of laboratory use.

*References:
- Vu, V., Barrett, J.C, Roycroft, J., Schuman, L., Dankovic, D., 1996. Workshop report: Chronic inhalation toxicity and carcinogenicity testing of respirable fibrous particles. Reg Tox Pharm 24, 202-212.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld, Germany)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: approx. 280 g for males and approx. 180 g for females
- Housing: housed in Makrolon (polycarbonate) cages type III with softwood (‘ssniff KB 8-15’) bedding material.
- Diet: commercial chow in pellet form (ssniff “V1534”) purchased from ssniff Spezialdiäten GmbH (Soest, Germany); ad libitum
- Water: tap water; ad libitum
- Acclimation period: Approx. one week the animals will be allowed to adjust and become acclimatised to the Fraunhofer ITEM environment. During the 2-3 weeks prior exposure start, all rats will be trained to the 6-hour restraint in nose-only tubes.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 55% ± 15%
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2020-08-04 To: 2021-02-17
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1.48 - <= 2.05 µm
Remarks on MMAD:
MMAD / GSD: please refer to Table 1 ('Any other information on materials and methods incl. tables')
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow-past nose-only inhalation exposure system
- Method of holding animals in test chamber: restrain tubes with a flexible stopper
- System of generating particulates/aerosols: The particulate sample aerosols were generated by dry dispersion with pressurized air. Cyclones (in line) were used to reduce the coarse moiety of the aerosol. For each nose-only exposure unit, the aerosol was generated by a high-pressure pneumatic disperser. The disperser was fed with the test/reference items under computerized control, i.e. with a feed back loop to the actual aerosol concentrations measured by an aerosol photometer. The photometer gives a scattering light signal which is proportional to the particle concentration, if the particle size distribution is constant. The ratio between photometer signal and concentration was determined throughout the study by comparing to gravimetric concentrations.
- Temperature, humidity, pressure in air chamber: Parameters were recorded by 20-minute means The were set at 22°C + 2°C for temperature and 55% + 15% for relative humidity.
- Air flow rate: 1 L/min
- Method of particle size determination: The MMAD was determined four times (once before exposure start and once per month during the exposure period for each test item exposure unit (3 units) by a cascade impactor (Marple impactor).
- Treatment of exhaust air: exhaled air is drawn off immediately by a cylinder surrounding the aerosol delivery cylinder

TEST ATMOSPHERE
- Brief description of analytical method used: Filter samples of the aerosols were taken daily to control the aerosol concentrations and to calibrate the aerosol photometers. The means are close to the target concentrations.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- see above ("Details on inhalation exposure")
- The target aerosol concentrations of 0.6, 2.5 and 10 mg/m³ Chromium Iron Oxide were achieved exactly, i.e. to 100% in each group.
Duration of treatment / exposure:
13 weeks (65 exposure days)
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
0.6 mg/m³ air (analytical)
Remarks:
SD: ± 0.09 mg/m³; 0.1 mg/lung (calculated total dose using MPPD v3.04)
Dose / conc.:
2.5 mg/m³ air (analytical)
Remarks:
SD: ± 0.35 mg/m³; 0.4 mg/lung (calculated total dose using MPPD v3.04)
Dose / conc.:
10.02 mg/m³ air (analytical)
Remarks:
SD: ± 1.49 mg/m³; 1.7 mg/lung (calculated total dose using MPPD v3.04)
No. of animals per sex per dose:
10+5 males and 10 females (1 day recovery); 5 males (28 days recovery); 5 males and females (90 days recovery) (total: 100 males and 60 females)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The concentrations were defined based on the preceding intratracheal instillation dose range finding (DRF A) study (Fraunhofer ITEM no. 02 N 20 502).
- Post-exposure recovery period: 1, 28, and 90 days

The nominal aerosol concentrations of 0.6, 2.5 and 10 mg/m³ were selected to achieve lung burden at the highest concentration that is at or above the lung overload conditions, i.e. impaired lung clearance. The test item deposition in the respiratory tract was modeled using the MPPD model (version 3.04), resulting in a deposited fraction of 4.7% (rel. density=5.1, MMAD/GSD=1.8 µm/1.5).
This deposited fraction was used to calculate the total deposited mass, using the following input parameters:
Morphometry: Semi-symmetric Long Evans
Example for deposited mass at 0.6 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 0.6 mg/m³ x 4.7% = 0.13 mg/lung
Example for deposited mass at 2.5 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 2.5 mg/m³ x 4.7% = 0.55 mg/lung
Example for deposited mass at 10 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 10 mg/m³ x 4.7% = 2.2 mg/lung
Retained mass at 10 mg/m³: approx. 1.7 mg/lung
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day (with the exception of weekends and public holidays: once daily)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before sacrifice

BODY WEIGHT: Yes
- Time schedule for examinations: 1 day before treatment and twice a week in the first 4 and once a week thereafter throughout the study for all animals

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
- Food consumption was determined for each group on a weekly basis.

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 (if drinking water study): Yes
- Water consumption was determined for each group on a weekly basis.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 1 day after the 90-day exposure period
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: 10 animals per sex and dose group
- Parameters examined: red blood cells (RBC), haemoglobin (HB), haematocrit (HCT), reticulocytes (RET), mean cell volume (MCV), mean haemoglobin/erythrocyte (MCH), mean haemoglobin concentration/erythrocyte (MCHC), prothrombin time (PT), thromboplastin time (TP), total white blood cells (WBC), differential white cell count (% and absolute), platelets (PTL)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 1 day after the 90-day exposure period
- Animals fasted: No
- How many animals: 10 animals per sex and dose group
- Parameters examined: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP), gamma-glutamyl transpeptidase (GGT), urea, triglycerides, total bilirubin, creatinine (CREA), total protein (TP), albumin (ALB), globulin (GLB), ALB/GLB, glucose (GLUC), cholesterol (CHOL), sodium (Na), calcium (Ca), potassium (K), phosphorous (P), chloride (CL)
URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: 1 and 90 days post-exposure
- Dose groups that were examined: all
- Number of animals: 5 (90 days post-exposure) - 10 (1 day post-exposure) per sex and dose group
- Parameters examined: total cell count, differential cell count (macrophages, neutrophils, eosinophils, lymphocytes; a total of 400 leukocytes per rat were evaluated), LDH, β-glucuronidase, and total protein

LUNG BURDEN: Yes (determination of lung:body weight ratio before and after treatment as well as chemical analysis)
- Time schedule for analysis: 1, 28, and 90 after the 90-day exposure period
- Dose groups that were examined: all
- Number of animals: 5 male rats
Sacrifice and pathology:
- rats were sacrificed 1, 28, and 90 days after the 90-day exposure period
- macroscopic examination: all animals were subjected to a complete necropsy
- organ weights were determined for the following organs: liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, thymus, spleen, brain, lung, and heart
- In the study the organs according to OECD guideline 413 were examined of the female and male animals of the clean air control and pigment 3 (Chromium iron oxide) high dose group after 90 days nose-only inhalation: adrenal glands, aorta, bone marrow (femur and sternum), brain (cerebrum, cerebellum, pons/medulla), coagulating glands, epididymis, esophagus, femur with knee joint, heart, intestine (duodenum, jejunum, ileum, cecum, colon, rectum), kidney, larynx, lung (left main lobe), liver, lymph nodes (lung-associated, mandibular, mesenteric), mammary gland, nasal cavity, olfactory bulb (in situ), ovaries, oviducts, pancreas, parathyroid glands, peripheral (sciatic) nerve, pituitary gland, prostate, salivary glands (mandibular, parotid, sublingual), seminal vesicles, skeletal muscle (biceps femoris), skin, spinal cord (cervical, thoracic and lumbar cords), spleen, stomach (forestomach and glandular stomach), testes, thymus, thyroid glands, trachea, urinary bladder, uterine cervix, uterus, vagina.
The following respiratory tract organs of animals 101-110 and 201-210 of group 1 and 4, respectively, were examined histopathologically: Nasal and Paranasal Cavities, pharynx (Laryngopharynx/Nasopharynx), larynx, trachea, lung, lung-associated lymph nodes (LALN).
- All organs were preserved and fixed in formalin at day 1 and 90 post-exposure. Histopathology was performed in 10 animals per sex of the control and high dose group at day 1 post-exposure.
Statistics:
Differences between groups will be considered statistically significant at p < 0.05. Data will be analysed using analysis of variance. If the group means differ significantly by the analysis of variance, the means of the treated groups will be compared with the means of the control groups using Dunnett’s test. The statistical evaluation of the histopathological findings will be done with the two-tailed Fisher test by the PROVANTIS system.
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:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
not specified
Description (incidence and severity):
Information will be added to the robust study summary upon availability
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- One day after the last exposure, enlarged lung-associated lymph nodes (LALN) were observed in 5 out of 20 male rats of the high dose group (vs. 1/20 in controls). This is a particle-specific lung clearance pathway.
- Lungs showed typical dose-dependent discolorations caused by the test item. On post-exposure day 1, the incidence for males was 0/20, 8/20, 18/20, 20/20 in the control, low, mid, and high dose group, respectively. On post-exposure day 90, the incidence for males was 0/5, 1/5, 5/5, 5/5 in the control, low, mid, and high dose group, respectively. On post-exposure day 1, the incidence for females was 1/10, 2/10, 9/10, 10/10 in the control, low, mid, and high dose group, respectively. On post-exposure day 90, the incidence for females was 0/5, 4/5, 4/4, 5/5 in the control, low, mid, and high dose group, respectively.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
High dose group:
- As exposure-related finding only an accumulation of particle-laden macrophages was observed in different parts of the respiratory tract.
- In the lung, a multifocal accumulation of particle-laden macrophages was seen in the alveoli in all male (all slight) and female (all slight) animals as well as in the bronchus-associated lymphoid tissue (BALT) in 9/10 males (8 very slight and 1 slight) and in all females (9 very slight and 1 slight). This lesion correlated with a macroscopically observed dark grey multifocal discoloration of up to 1mm in diameter in the lung.
- In the nasal cavity, a very slight multifocal accumulation of particle-laden macrophages was found in 3/10 males and 3/10 females in the nose-associated lymphoid tissue (NALT).
- In the lung-associated lymph nodes (LALN), there was a multifocal very slight accumulation of particle-laden macrophages in 7/10 males and in 5/10 females. This lesion correlated with a macroscopically observed enlargement in few animals.
- The accumulation of particle-laden macrophages was not associated with further pathologic lesions in any organ.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
MORTALITY:
- Due to a trauma to the tail, one animal (female # 3202; mid dose group) was sacrificed preterminally. The trauma was diagnosed as an acute fracture of the tail. The lesion was interpreted to be incidental without any relation to the exposure.

ORGAN WEIGHT FINDINGS INCL ORGAN / BODY WEIGHT RATIOS:
- At day-92 after the last exposure, females of the high dose group showed statistically significantly increased absolute and relative weights of the left (+39% and +39.9%, respectively) and right ovary (+36.5% and +37.2%, respectively), when compared to the vehicle control group. The effect showed, however, no clear dose-response relationship and was without a histopathological correlate. Thus, the finding is considered to be not biologically relevant.
- At day 92 after the last exposure, males of the high dose group showed a statistically significantly increased relative thymus weight (+42.2%). The absolute weight was, however, not statistically significantly altered. Moreover, no such effect was observed in females and the finding was without a histopathological correlate, and thus, considered to be not biologically relevant.

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC:
- Clean air control: In one animal of the control group, a lymphoma of the hematolymphoid system with infiltration of lymphoma cells into the bone marrow, liver, lung-associated, mandibular and mesenteric lymph nodes, and spleen was observed. This correlated with macroscopically observed enlarged spleen and lung-associated lymph nodes. In addition, single lesions were seen in different other organs, which represented commonly found background lesions in this rat strain.
- All the other findings within the different organs occurred in single animals and were interpreted to be incidental without any relation to the exposure.

BALF ANALYSES:
- At days 1 and 90 (females only) post-exposure no statistically significant increases of polymorphonuclear neutrophils were detected in any treatment group. The PMN percentages (in the range from 2.9% to 5.9%, males - 0.7% to 1.9%, females) are close to historical clean air control data; thus, the test item did not induce a relevant PMN-related lung inflammation. Lymphocyte levels were also found low and at control levels (<1%).
- For lactic dehydrogenase, ß-glucuronidase and total protein, no relevant statistically significant increases were detected in any group of both sexes and sacrifice dates.
- Lung weights: In both sexes, no statistically significant changes as compared to concurrent controls.

LUNG BURDEN ANALYSIS:
- The lung burden analysis did not show any statistically significantly increase in the lung to bodyweight ratio at post-exposure day-1 and 92, when compared to the vehicle control group.
- The results of the chemical lung burden analysis will be submitted later.
Key result
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Conclusions:
In this 90-day repeated dose toxicity by inhalation study, rats were exposed via nose-only inhalation towards aerosol concentrations of 0.6, 2.5 and 10mg/m³ of chromium iron oxide.
All animals (but one due to accident) survived the test period and were euthanized at scheduled dates. Effects indicating systemic toxicity were not observed. Sex-specific differences were not detected.
No relevant statistically significant changes as compared to concurrent controls were observed for: body weight and body weight development, food and water consumption, haematology, lung weights, bronchoalveolar lavage fluid (BALF) analysis, histopathological evaluation.
Some adaptive exposure-related findings were detected in the investigated high dose group (Pigment 3 - Chromium iron oxide) within the lung, the nasal cavity, and the lung-associated lymph nodes: accumulation of particle-laden macrophages, which was not associated with further pathologic lesions in any organ. The lesion occurred statistically significant in the lung and in the lung-associated lymph nodes. This lesion is considered to represent a non-adverse adaptive change.
Due to an absence of any adverse findings the NOAEC is considered to be above 10 mg/m³, the maximum tolerated concentration based on lung burden calculations.


The measures implemented during the ongoing Covid-19 pandemic lead to a delay in laboratory capacities and delays in the study conduct. Due to this, the following parameters are not yet available and will be added to the robust study summary upon availability:
(i) chemical analysis of lung burden
(ii) clinical chemistry
(iii) QAU evaluation and study finalisation
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
start: 2020-07-30 and still ongoing
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study
Version / remarks:
2018-06-25
Deviations:
yes
Remarks:
Ophthalmology not performed (this endpoint is not sensitive in particle studies); urine analysis not performed (endpoint optional only in guideline)
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP certificate signed 2018-11-22
Limit test:
no
Species:
rat
Strain:
other: Crl:WI (Han)
Details on species / strain selection:
Wistar rats are commonly used in subchronic and chronic inhalation toxicity studies. They fulfil the criteria stated by a U.S. EPA Workshop (Vu et al., 1996)* such as (i) a low background rate of neoplasia, (ii) a low background rate of pulmonary disease, (iii) longevity, and (iv) a history of laboratory use.

*References:
- Vu, V., Barrett, J.C, Roycroft, J., Schuman, L., Dankovic, D., 1996. Workshop report: Chronic inhalation toxicity and carcinogenicity testing of respirable fibrous particles. Reg Tox Pharm 24, 202-212.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld, Germany)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: approx. 280 g for males and approx. 180 g for females
- Housing: housed in Makrolon (polycarbonate) cages type III with softwood (‘ssniff KB 8-15’) bedding material.
- Diet: commercial chow in pellet form (ssniff “V1534”) purchased from ssniff Spezialdiäten GmbH (Soest, Germany); ad libitum
- Water: tap water; ad libitum
- Acclimation period: Approx. one week the animals will be allowed to adjust and become acclimatised to the Fraunhofer ITEM environment. During the 2-3 weeks prior exposure start, all rats will be trained to the 6-hour restraint in nose-only tubes.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 55% ± 15%
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2020-08-04 To: 2021-02-17
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1.48 - <= 2.05 µm
Remarks on MMAD:
MMAD / GSD: please refer to Table 1 ('Any other information on materials and methods incl. tables')
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow-past nose-only inhalation exposure system
- Method of holding animals in test chamber: restrain tubes with a flexible stopper
- System of generating particulates/aerosols: The particulate sample aerosols were generated by dry dispersion with pressurized air. Cyclones (in line) were used to reduce the coarse moiety of the aerosol. For each nose-only exposure unit, the aerosol was generated by a high-pressure pneumatic disperser. The disperser was fed with the test/reference items under computerized control, i.e. with a feed back loop to the actual aerosol concentrations measured by an aerosol photometer. The photometer gives a scattering light signal which is proportional to the particle concentration, if the particle size distribution is constant. The ratio between photometer signal and concentration was determined throughout the study by comparing to gravimetric concentrations.
- Temperature, humidity, pressure in air chamber: Parameters were recorded by 20-minute means The were set at 22°C + 2°C for temperature and 55% + 15% for relative humidity.
- Air flow rate: 1 L/min
- Method of particle size determination: The MMAD was determined four times (once before exposure start and once per month during the exposure period for each test item exposure unit (3 units) by a cascade impactor (Marple impactor).
- Treatment of exhaust air: exhaled air is drawn off immediately by a cylinder surrounding the aerosol delivery cylinder

TEST ATMOSPHERE
- Brief description of analytical method used: Filter samples of the aerosols were taken daily to control the aerosol concentrations and to calibrate the aerosol photometers. The means are close to the target concentrations.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- see above ("Details on inhalation exposure")
- The target aerosol concentrations of 0.6, 2.5 and 10 mg/m³ Chromium Iron Oxide were achieved exactly, i.e. to 100% in each group.
Duration of treatment / exposure:
13 weeks (65 exposure days)
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
0.6 mg/m³ air (analytical)
Remarks:
SD: ± 0.09 mg/m³; 0.1 mg/lung (calculated total dose using MPPD v3.04)
Dose / conc.:
2.5 mg/m³ air (analytical)
Remarks:
SD: ± 0.35 mg/m³; 0.4 mg/lung (calculated total dose using MPPD v3.04)
Dose / conc.:
10.02 mg/m³ air (analytical)
Remarks:
SD: ± 1.49 mg/m³; 1.7 mg/lung (calculated total dose using MPPD v3.04)
No. of animals per sex per dose:
10+5 males and 10 females (1 day recovery); 5 males (28 days recovery); 5 males and females (90 days recovery) (total: 100 males and 60 females)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The concentrations were defined based on the preceding intratracheal instillation dose range finding (DRF A) study (Fraunhofer ITEM no. 02 N 20 502).
- Post-exposure recovery period: 1, 28, and 90 days

The nominal aerosol concentrations of 0.6, 2.5 and 10 mg/m³ were selected to achieve lung burden at the highest concentration that is at or above the lung overload conditions, i.e. impaired lung clearance. The test item deposition in the respiratory tract was modeled using the MPPD model (version 3.04), resulting in a deposited fraction of 4.7% (rel. density=5.1, MMAD/GSD=1.8 µm/1.5).
This deposited fraction was used to calculate the total deposited mass, using the following input parameters:
Morphometry: Semi-symmetric Long Evans
Example for deposited mass at 0.6 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 0.6 mg/m³ x 4.7% = 0.13 mg/lung
Example for deposited mass at 2.5 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 2.5 mg/m³ x 4.7% = 0.55 mg/lung
Example for deposited mass at 10 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 10 mg/m³ x 4.7% = 2.2 mg/lung
Retained mass at 10 mg/m³: approx. 1.7 mg/lung
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day (with the exception of weekends and public holidays: once daily)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before sacrifice

BODY WEIGHT: Yes
- Time schedule for examinations: 1 day before treatment and twice a week in the first 4 and once a week thereafter throughout the study for all animals

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
- Food consumption was determined for each group on a weekly basis.

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 (if drinking water study): Yes
- Water consumption was determined for each group on a weekly basis.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 1 day after the 90-day exposure period
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: 10 animals per sex and dose group
- Parameters examined: red blood cells (RBC), haemoglobin (HB), haematocrit (HCT), reticulocytes (RET), mean cell volume (MCV), mean haemoglobin/erythrocyte (MCH), mean haemoglobin concentration/erythrocyte (MCHC), prothrombin time (PT), thromboplastin time (TP), total white blood cells (WBC), differential white cell count (% and absolute), platelets (PTL)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 1 day after the 90-day exposure period
- Animals fasted: No
- How many animals: 10 animals per sex and dose group
- Parameters examined: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP), gamma-glutamyl transpeptidase (GGT), urea, triglycerides, total bilirubin, creatinine (CREA), total protein (TP), albumin (ALB), globulin (GLB), ALB/GLB, glucose (GLUC), cholesterol (CHOL), sodium (Na), calcium (Ca), potassium (K), phosphorous (P), chloride (CL)
URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: 1 and 90 days post-exposure
- Dose groups that were examined: all
- Number of animals: 5 (90 days post-exposure) - 10 (1 day post-exposure) per sex and dose group
- Parameters examined: total cell count, differential cell count (macrophages, neutrophils, eosinophils, lymphocytes; a total of 400 leukocytes per rat were evaluated), LDH, β-glucuronidase, and total protein

LUNG BURDEN: Yes (determination of lung:body weight ratio before and after treatment as well as chemical analysis)
- Time schedule for analysis: 1, 28, and 90 after the 90-day exposure period
- Dose groups that were examined: all
- Number of animals: 5 male rats
Sacrifice and pathology:
- rats were sacrificed 1, 28, and 90 days after the 90-day exposure period
- macroscopic examination: all animals were subjected to a complete necropsy
- organ weights were determined for the following organs: liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, thymus, spleen, brain, lung, and heart
- In the study the organs according to OECD guideline 413 were examined of the female and male animals of the clean air control and pigment 3 (Chromium iron oxide) high dose group after 90 days nose-only inhalation: adrenal glands, aorta, bone marrow (femur and sternum), brain (cerebrum, cerebellum, pons/medulla), coagulating glands, epididymis, esophagus, femur with knee joint, heart, intestine (duodenum, jejunum, ileum, cecum, colon, rectum), kidney, larynx, lung (left main lobe), liver, lymph nodes (lung-associated, mandibular, mesenteric), mammary gland, nasal cavity, olfactory bulb (in situ), ovaries, oviducts, pancreas, parathyroid glands, peripheral (sciatic) nerve, pituitary gland, prostate, salivary glands (mandibular, parotid, sublingual), seminal vesicles, skeletal muscle (biceps femoris), skin, spinal cord (cervical, thoracic and lumbar cords), spleen, stomach (forestomach and glandular stomach), testes, thymus, thyroid glands, trachea, urinary bladder, uterine cervix, uterus, vagina.
The following respiratory tract organs of animals 101-110 and 201-210 of group 1 and 4, respectively, were examined histopathologically: Nasal and Paranasal Cavities, pharynx (Laryngopharynx/Nasopharynx), larynx, trachea, lung, lung-associated lymph nodes (LALN).
- All organs were preserved and fixed in formalin at day 1 and 90 post-exposure. Histopathology was performed in 10 animals per sex of the control and high dose group at day 1 post-exposure.
Statistics:
Differences between groups will be considered statistically significant at p < 0.05. Data will be analysed using analysis of variance. If the group means differ significantly by the analysis of variance, the means of the treated groups will be compared with the means of the control groups using Dunnett’s test. The statistical evaluation of the histopathological findings will be done with the two-tailed Fisher test by the PROVANTIS system.
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:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
not specified
Description (incidence and severity):
Information will be added to the robust study summary upon availability
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- One day after the last exposure, enlarged lung-associated lymph nodes (LALN) were observed in 5 out of 20 male rats of the high dose group (vs. 1/20 in controls). This is a particle-specific lung clearance pathway.
- Lungs showed typical dose-dependent discolorations caused by the test item. On post-exposure day 1, the incidence for males was 0/20, 8/20, 18/20, 20/20 in the control, low, mid, and high dose group, respectively. On post-exposure day 90, the incidence for males was 0/5, 1/5, 5/5, 5/5 in the control, low, mid, and high dose group, respectively. On post-exposure day 1, the incidence for females was 1/10, 2/10, 9/10, 10/10 in the control, low, mid, and high dose group, respectively. On post-exposure day 90, the incidence for females was 0/5, 4/5, 4/4, 5/5 in the control, low, mid, and high dose group, respectively.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
High dose group:
- As exposure-related finding only an accumulation of particle-laden macrophages was observed in different parts of the respiratory tract.
- In the lung, a multifocal accumulation of particle-laden macrophages was seen in the alveoli in all male (all slight) and female (all slight) animals as well as in the bronchus-associated lymphoid tissue (BALT) in 9/10 males (8 very slight and 1 slight) and in all females (9 very slight and 1 slight). This lesion correlated with a macroscopically observed dark grey multifocal discoloration of up to 1mm in diameter in the lung.
- In the nasal cavity, a very slight multifocal accumulation of particle-laden macrophages was found in 3/10 males and 3/10 females in the nose-associated lymphoid tissue (NALT).
- In the lung-associated lymph nodes (LALN), there was a multifocal very slight accumulation of particle-laden macrophages in 7/10 males and in 5/10 females. This lesion correlated with a macroscopically observed enlargement in few animals.
- The accumulation of particle-laden macrophages was not associated with further pathologic lesions in any organ.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
MORTALITY:
- Due to a trauma to the tail, one animal (female # 3202; mid dose group) was sacrificed preterminally. The trauma was diagnosed as an acute fracture of the tail. The lesion was interpreted to be incidental without any relation to the exposure.

ORGAN WEIGHT FINDINGS INCL ORGAN / BODY WEIGHT RATIOS:
- At day-92 after the last exposure, females of the high dose group showed statistically significantly increased absolute and relative weights of the left (+39% and +39.9%, respectively) and right ovary (+36.5% and +37.2%, respectively), when compared to the vehicle control group. The effect showed, however, no clear dose-response relationship and was without a histopathological correlate. Thus, the finding is considered to be not biologically relevant.
- At day 92 after the last exposure, males of the high dose group showed a statistically significantly increased relative thymus weight (+42.2%). The absolute weight was, however, not statistically significantly altered. Moreover, no such effect was observed in females and the finding was without a histopathological correlate, and thus, considered to be not biologically relevant.

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC:
- Clean air control: In one animal of the control group, a lymphoma of the hematolymphoid system with infiltration of lymphoma cells into the bone marrow, liver, lung-associated, mandibular and mesenteric lymph nodes, and spleen was observed. This correlated with macroscopically observed enlarged spleen and lung-associated lymph nodes. In addition, single lesions were seen in different other organs, which represented commonly found background lesions in this rat strain.
- All the other findings within the different organs occurred in single animals and were interpreted to be incidental without any relation to the exposure.

BALF ANALYSES:
- At days 1 and 90 (females only) post-exposure no statistically significant increases of polymorphonuclear neutrophils were detected in any treatment group. The PMN percentages (in the range from 2.9% to 5.9%, males - 0.7% to 1.9%, females) are close to historical clean air control data; thus, the test item did not induce a relevant PMN-related lung inflammation. Lymphocyte levels were also found low and at control levels (<1%).
- For lactic dehydrogenase, ß-glucuronidase and total protein, no relevant statistically significant increases were detected in any group of both sexes and sacrifice dates.
- Lung weights: In both sexes, no statistically significant changes as compared to concurrent controls.

LUNG BURDEN ANALYSIS:
- The lung burden analysis did not show any statistically significantly increase in the lung to bodyweight ratio at post-exposure day-1 and 92, when compared to the vehicle control group.
- The results of the chemical lung burden analysis will be submitted later.
Key result
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Conclusions:
In this 90-day repeated dose toxicity by inhalation study, rats were exposed via nose-only inhalation towards aerosol concentrations of 0.6, 2.5 and 10mg/m³ of chromium iron oxide.
All animals (but one due to accident) survived the test period and were euthanized at scheduled dates. Effects indicating systemic toxicity were not observed. Sex-specific differences were not detected.
No relevant statistically significant changes as compared to concurrent controls were observed for: body weight and body weight development, food and water consumption, haematology, lung weights, bronchoalveolar lavage fluid (BALF) analysis, histopathological evaluation.
Some adaptive exposure-related findings were detected in the investigated high dose group (Pigment 3 - Chromium iron oxide) within the lung, the nasal cavity, and the lung-associated lymph nodes: accumulation of particle-laden macrophages, which was not associated with further pathologic lesions in any organ. The lesion occurred statistically significant in the lung and in the lung-associated lymph nodes. This lesion is considered to represent a non-adverse adaptive change.
Due to an absence of any adverse findings the NOAEC is considered to be above 10 mg/m³, the maximum tolerated concentration based on lung burden calculations.


The measures implemented during the ongoing Covid-19 pandemic lead to a delay in laboratory capacities and delays in the study conduct. Due to this, the following parameters are not yet available and will be added to the robust study summary upon availability:
(i) chemical analysis of lung burden
(ii) clinical chemistry
(iii) QAU evaluation and study finalisation
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Animal data - oral route:


A 28-day repeated dose toxicity study (LPT, 2018) was conducted in rats as a limit test to assess the effect of the pigment on rats following repeated oral administration. The study was performed according to OECD test guideline 407 and in compliance with GLP.


Male and female rats were administered with the pigment by oral gavage for 28 days at a dose of 1000 mg/kg bw/day in 0.8% aqueous hydroxyl propyl methylcellulose gel. A concurrent control group was administered untreated vehicle.


No clinical signs of toxicity were observed, and no animals died during the administration period. No changes in bodyweight gains, food consumption, haematology, clinical chemistry, organ weights or macropathology were observed which could be attributed to treatment with the test compound. The histomorphological examination of rat organs did not reveal any morphological lesions attributable to the administration of the test item. There were no morphological differences between the control rats and the test item-treated animals. No adverse effects were observed on the male and female reproductive organs.


Furthermore, individual 24-hour urine samples were collected from all animals after the last dosing prior to the scheduled sacrifice, and in addition plasma samples were collected from each animal at the day of sacrifice.The plasma and urine samples were analysed for total chromium and iron content. The comparison of the total administered final pigment dose of 1000mg/kg bw with the total mean Cr and Fe content recovered in 24-urine samples, as calculated from the mean 24h-urine collection volumes of 12.1 ml for males and of 8.5 ml for females, would correspond to a total bioavailable chromium fraction of 0.0022% for males and <0.0001% for females, and a total bioavailable iron fraction of 0.00005% for males and 0.00002 for females (under the simplified assumption that excretion of both elements occurs to a relevant extent via urine which is the case for chromium, whereas for iron excretion via faeces also plays a role (1)). The Cr and Fe concentrations of plasma samples, collected from control and dose group animals at the day of sacrifice, were below 0.0006 µg and 0.0013 µg Cr and below 0.24 µg and 0.20 µg Fe/L plasma.


It is concluded that the pigment was well tolerated and that no signs of systemic toxicity whatsoever were observed in rats when administered at a dose of 1000 mg/kg bw/day for up to 28 days. Either no or only marginal increases in Cr and Fe plasma concentrations were observed, and only a minor fraction (<0.002%) of the total administered dose of Cr and Fe was collected via urine, documenting the lack of bioavailability of this pigment. The no observed adverse effect level (NOAEL) in rats is 1000 mg/kg/day.


 


1: Excretion of iron predominantly occurs via faeces, although trace amounts of iron are also excreted via urine, desquamated gastrointestinal cells, and bile(INACG, 1993; IOM, 2001; EFSA, 2004; EVM, 2003, Gordeuk et al., 1986; Huebers et al., 1986;Lopez and Cámara Martos, 2004; Teucher et al., 2004). Most chromium(III) is cleared rapidly from the blood and excreted in the urine with small amounts also being lost in perspiration, bile and faeces (Anderson et al., 1997b; Gargas et al. 1994; Jeejeebhoy, 1999; ATSDR, 2000; IOM, 2001; Hepburn and Vincent, 2002).


 


Animal data - inhalation route:


In a 90 -day repeated dose toxicity by inhalation study, rats were exposed via nose-only inhalation towards aerosol concentrations of 0.6, 2.5 and 10 mg/m³ of chromium iron oxide.


All animals (but one due to accident) survived the test period and were euthanized at scheduled dates. Effects indicating systemic toxicity were not observed. Sex-specific differences were not detected.


No relevant statistically significant changes as compared to concurrent controls were observed for: body weight and body weight development, food and water consumption, haematology, lung weights, bronchoalveolar lavage fluid (BALF) analysis, histopathological evaluation.


Some adaptive exposure-related findings were detected in the investigated high dose group (Pigment 3 - Chromium iron oxide) within the lung, the nasal cavity, and the lung-associated lymph nodes: accumulation of particle-laden macrophages, which was not associated with further pathologic lesions in any organ. The lesion occurred statistically significant in the lung and in the lung-associated lymph nodes. This lesion is considered to represent a non-adverse adaptive change.


Due to an absence of any adverse findings the NOAEC is considered to be above 10 mg/m³, the concentration at or above the onset of lung overload , thus constituting the maximum tolerated concentration.


 


 


The measures implemented during the ongoing Covid-19 pandemic lead to a delay in laboratory capacities and delays in the study conduct (see attached documentation in the study record for further information). Due to this, the following parameters are not yet available and will be added to the robust study summary upon availability:


(i) chemical analysis of lung burden


(ii) clinical chemistry


(iii) QAU evaluation and study finalisation


 

Justification for classification or non-classification

No signs of systemic toxicity whatsoever were observed in rats when administered at a dose of 1000 mg/kg bw/day for up to 28 days and after 90 -days nose-only inhalation up to the maximum tolerated concentration. No classification for Specific Target Organ Toxicity-Repeated Exposure (STOT-RE) according to EC Regulation No. 1272/2008 is required.