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Description of key information

Sub-acute oral toxicity

28 -day repeated dose toxicity studies were conducted in rats as a limit test to assess the effect of the pigments chromium iron oxide and manganese alumina pink corundum on rats following repeated oral administration. The studies were performed according to OECD test guideline 407 and in compliance with GLP.

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.The no observed adverse effect level (NOAEL) in rats is 1000 mg/kg/day.

Sub-chronic oral toxicity

The low order of repeated dose toxicity of Hematite chromium green black is manifested in (i) an absence of a concern for the endpoint repeated dose toxicity for the soluble and insoluble forms of the metals present in Hematite chromium green black, (ii) the very clear finding that Hematite chromium green black is of poor bioavailability in a wide range of physiological media (iii) Hematite chromium green black shows no effects in a number of in vitro and in vivo toxicological tests and (iv) the physico-chemical properties demonstrate the Hematite chromium green black is of general inertness (point iii and iv please refer to the respective waiver). Furthermore, concerning this minimal fraction of dose that is available for systemic absorption, the inorganic nature of the metals present in Hematite chromium green black precludes any form of metabolism in mammals.

 

Sub-chronic inhalation toxicity

The absence of repeated dose toxicity via the inhalation route of Hematite chromium green black is deduced from (i) an absence of concern for the endpoint repeated dose toxicity via the inhalation route for the oxidic forms of the metals present in Hematite chromium green black, (ii) the low inhalability of hematite chromium green black by rats (based on MPPD modelling), (iii) the very clear finding that Hematite chromium green black is of poor bioavailability in a wide range of physiological media, (iv) Hematite chromium green black shows no effects in a number of in vitro and in vivo toxicological tests, and (v) the PSLT properties of Hematite chromium green black.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-01-14 to 2015-02-11
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: 136.4 g - 158.6 g; females: 132.8 g - 146.1 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: 7 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 nos.: 12G23-N03 and 13D03-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-150/6-27/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-150/6-27). 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.
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). Aluminium was detected at the wavelength 167.019 nm, 308.215 nm, 394.401 nm and 396.152 nm; manganese was detected at the wavelength 257.610 nm, 259.372 nm, 260.568 nm, 293.305 nm, 293.931 nm and 294.921 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 0.999928. 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:
Al: 167.019 nm, 308.215 nm, 394.401 nm and 396.152 nm
Mn: 257.610 nm, 259.372 nm, 260.568 nm, 293.305 nm, 293.931 nm and 294.921 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: 0.096 – 0.118 µg/L (Al); 0.036 – 0.062 µg/L (Mn)
- LOQ: 0.289 – 0.355 µg/L (Al);0.109 – 0.187 µg/L ((Mn)
- correlation coefficient: 0.999928 (Al); 0.999930 (Mn)

The certified reference material TMDA-52.4, TMDA-54.2 and TMDA-70.2 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 respectivecertified value. Selected samples were fortified with a known amount of aluminium and manganese (by standard addition of commercial standards) to determine the standard recovery of aluminium and manganese. Data are compiled in Table 4 - 5. For fortified samples, recoveries were 101 - 107% for Al and 100 - 103% for Mn.

Results:
Dose verification:
nominal dose: 1,000 mg/kg bw pigment (468 mg/kg bw Al, 56 mg/kg bw Mn)
Results:
Analysis of stability and concentration (3 samples):
Recovery [%]:
Al: 88.7 - 106
Mn: 88.6 - 102

Anaylsis of homogenity (3samples):
Recovery [%]:
Al: 82.1-91.3
Mn: 81.8 - 90.6

Anaylsis of concentration (1sample):
Recovery [%]:
Al: 91.7
Mn: 92.5
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-150/6-27/y)
Urine and plasma samples were obtained at study termination. Urine and plasma samples were analysed for aluminium and manganese 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:
mortality observed, non-treatment-related
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, external appearance or faeces were noted for the male and female rats treated with 1000 mg manganese alumina pink corundum/kg bw/day or for the animals treated with the vehicle control.
- faeces of the control and test item-treated animals were formed normally.

MORTALITY
- no test item-related deaths occurred.
- 1/5 male control animals died prematurely during blood withdrawal for laboratory examinations (not a test item-related finding; death caused by stress during blood withdrawal and anaesthesia).

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 manganese alumina pink corundum/kg b.w./day (data 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 test item/kg bw/day compared to the control group.
- statistically significant differences (p ≤ 0.05) in relative food consumption of test item-treated animals compared to the control animals were recorded (no test item-related findings):
males (test week 4): increased relative food consumption

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 test item/kg bw/day or for the animals treated with the vehicle control.
- no pathological changes were noted on the adnexa oculi, conjunctiva, cornea, anterior chamber, iris (pupil dilated), lens, vitreous body and fundus.

HAEMATOLOGICAL FINDINGS
- no test item-related influence in haematological and coagulation parameters was noted for the male and female rats treated with 1000 mg manganese alumina pink corundum/kg bw/day compared to the control group.
- statistically significant differences (p ≤ 0.05) in haematological parameters of test item-treated animals compared to the control animals were recorded (no test item-related findings):
males (test day 29): decreased erythrocytes (control group: 8.394 ± 0.330 x10E6/µL vs. treatment group: 7.894 ± 0.297 x10E6/µL) and increased mean corpuscular volume (control group: 57.80 ± 0.70 fL vs. treatment group: 60.24 ± 1.96 fL)
females (test day 29): decreased platelets (control group: 1071.6 ± 153.2 x10³/µL vs. treatment group: 874.0 ± 101.0 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 test item/kg bw/day compared to the control group.
- statistically significant differences in biochemical parameters of test item-treated animals compared to the control animals were recorded (no test item-related findings):
males (test day 29; p ≤ 0.01 and p ≤ 0.05): increased creatinine (control group: 36.0 ± 2.0 µmol/L vs. treatment group: 39.8 ± 1.1 µmol/L) and increased chloride (control group: 100.0 ± 1.4 mmol/L vs. treatment group: 101.8 ± 0.8 mmol/L)
females (test day 29; p ≤ 0.05): increased calcium (control group: 2.570 ± 0.027 mmol/L vs. treatment group: 2.688 ± 0.083 mmol/L) and increased cholesterol (control group: 1.854 ± 0.123 mmol/L vs. treatment group: 2.236 ± 0.321 mmol/L)
However, the stated clinical chemistry 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, although the values for the spontaneous motility appeared to be below the normal range.
- statistically significant differences in neurological parameters of test item-treated animals compared to the control animals were recorded (no test item-related findings):
males (test week 4; p ≤ 0.01): increased spontaneous motility*
females (test week 4; p ≤ 0.01 and p ≤ 0.05): increased hindlimb grip strength and increased spontaneous motility*
* effect is due to the relative low and high value observed for the control group

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 test item/kg bw/day compared to the control group.
- statistically significant differences in organ weights of test item-treated animals compared to the control animals were recorded (no test item-related findings):
males (test day 29; p ≤ 0.05): decreased absolute brain weight (control group: 1.996 ± 0.064 g vs. treatment group: 1.924 ± 0.021 g), relative kidney weight (left)(control group: 4.904 ± 0.286 g/kg bw vs. treatment group: 4.406 ± 0.274 g/kg bw), and relative kidney weight (right)(control group: 5.042 ± 0.283 g/kg bw vs. treatment group: 4.529 ± 0.234 g/kg bw)
females (test day 29; p ≤ 0.01): decreased relative adrenal weight (right)(control group: 0.2124 ± 0.0274 g/kg bw vs. treatment group: 0.1553 ± 0.0194 g/kg bw) and absolute adrenal weight (right)(control group: 0.0422 ± 0.0055 g vs. treatment group: 0.0306 ± 0.0036 g)
However, the reported organ weights are within the normal range 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.

GROSS PATHOLOGICAL FINDINGS
- none of the male and female rats treated with 1000 mg manganese alumina pink corundum/kg bw/day revealed any macroscopic changes at necropsy on test day 29.

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 control and treatment groups).
- inflammatory lesions occurred in various organs such as liver, trachea, larynx, kidney, epididymis, and prostate in both control and test item-treated animals. The inflammatory reaction was associated with a normal lymphoid hyperplasia in the spleen, lymph nodes and the gut-associated lymphoid tissue in the intestine.
- the testis, epididymis, prostate and seminal vesicle of the control and test item-treated rats showed an age-related normal morphology. There was no difference between the control group and treatment group. The mild to moderate atrophy of the germinative epithelium with increase of giant cells and an increase of immature cells in the canaliculi of the epididymis in 2 control animals was a coincidental finding.
- a minimal to mild reduction of lymphoid tissue (involution) was noted in the cortex and medulla of the thymus in the male and female rats of the control group and the treatment group. There was no difference between the control and treatment groups (findings corresponded in type, incidence and severity to the age of the animals).
- a minimal to mild single cell or peripheral fatty infiltrations in the hepatocytes and a minimal fatty infiltration in the tubular epithelial cells of the kidney were observed in control and test item-treated animals. There were no differences between the control group and treatment group (findings were within physiological limits).
Coincidental findings in a small number of control- and test animals are:
Epididymis: increase of immature cells in controls;
Larynx: glandular ectasia;
Kidney: basophilic tubular cells;
Mammary glands: glandular hyperplasia particularly in male rat;
Testis: atrophy of the germinative epithelium with giant cells;
Thyroid: squamous cell cyst;
Trachea: tracheal gland dilatation;
Urinary bladder: proteinaceous content in male rats;
Uterus: hydrometra.
There was no difference between the control group and the treatment group in male and female rats.
Please also refer to the field "Attached background material" below.

TOXICOKINETICS
Manganese and aluminium are of negligible bioavailability from the test substance Manganese alumina pink corundum: by recalculating the urine levels and setting them into relation to the administered dose of the individual elements Mn and Al, 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 manganese alumina pink corundum/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 manganese and aluminium during a 24 hour urine and plasma sampling period was demonstrated to be negligible considering that <<0.002% 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 all two elements are not biologically available upon ingestion of the pigment Manganese alumina pink corundum
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Based on the explanation given in the read-across assessment framework document (attached in IUCLID section 13) an analogue approach for read-across of the endpoint “28d repeated dose toxicity: oral” from the structural analogues to the target substance is considered justified.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Source substance 1: no mortality observed
Source substance 2: mortality observed, non-treatment related
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
Key result
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Conclusions:
NOAEL (oral; rats) > 1000 mg test item/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.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

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

Sub-acute oral toxicity

Substance-specific information on repeated dose toxicity of hematite, chromium green black is not available. Hence, read-across from the source substances chromium iron oxide and manganese alumina pink corundum in an analogue approach was done. For further description please refer to the read -acrosss assessment framework document in IUCLID section 13.

28 -day repeated dose toxicity studies were conducted in rats as a limit test to assess the effect of the pigments chromium iron oxide and manganese alumina pink corundum on rats following repeated oral administration. The studies were performed according to OECD test guideline 407 and in compliance with GLP.

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 compounds. The histomorphological examination of rat organs did not reveal any morphological lesions attributable to the administration of the test items. 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.

Sub-chronic oral toxicity

The low order of repeated dose toxicity of Hematite chromium green black is manifested in (i) an absence of a concern for the endpoint repeated dose toxicity for the soluble and insoluble forms of the metals present in Hematite chromium green black, (ii) the very clear finding that Hematite chromium green black is of poor bioavailability in a wide range of physiological media (iii) Hematite chromium green black shows no effects in a number of in vitro and in vivo toxicological tests and (iv) the physico-chemical properties demonstrate the Hematite chromium green black is of general inertness (point iii and iv please refer to the respective waiver). Furthermore, concerning this minimal fraction of dose that is available for systemic absorption, the inorganic nature of the metals present in Hematite chromium green black precludes any form of metabolism in mammals.

 

Overall, the conduct of a repeated dose toxicity study cannot be expected to contribute any relevant information to the assessment of information on repeated dose toxicity. As a result, the need for such testing is waived in accordance with regulation (EC) 1907/2008, Annex XI, Section 1.2. Based on the weight-of-evidence information for Hematite chromium green black it is concluded that Hematite chromium green black does not present a repeated dose toxicity hazard, as follows:

 

(i) Supporting information for repeated dose toxicity with metals contained in Hematite chromium green black

 

Chromium

In a 90-day repeated dose toxicity study conducted by Ivankovic & Preussmann (1975) administration of 5% chromium oxide green (equivalent to ~2800 mg/kg bw/day in males and ~2500 mg/kg bw/day in females) via the diet to rats did not produce signs of toxicity and no detectable differences from untreated controls. Food intake was normal throughout the study and body weight gain was comparable to control rats. No adverse findings were noted in haematology and clinical biochemistry and urinalysis war normal. No macroscopic changes in gross pathology were observed. With the exception of the weights or the spleen and liver, which showed some reduction on the treated animals, there were no macroscopic or histological changes. The only effect observed was that the faeces of treated animals showed an intense green colouration, which indicated significant excretion of the administered pigment. Thus, dietary administration of 5% or lower of chromium oxide caused no significant toxic effect in male and female rats.

 

Further on, in 2014 the EFSA CONTAM Panel concluded: “In general, Cr(III) displays very little to no toxicity in animals up to the highest dose tested. In the Shara et al. (2007) study, the only effects observed in rats dietary exposed to 25 mg/kg chromium nicotinate were statistically significant decreases in body weight gain, between 5.5 and 14.9 %, which were not considered as adverse by the CONTAM Panel. The lack of toxicity observed at high concentrations of Cr(III) may reflect the poor absorption of Cr(III) by the oral route of exposure. The use of baked bread as a vehicle may have further reduced the absorption of chromium in the Ivankovic and Preussman (1975) study.

The studies conducted by the National Toxicology Program (NTP, 2010) have been performed according to present scientific guidance and reported a comprehensive assessment of toxicological endpoints and adequate reporting for estimation of doses in mg Cr(III)/kg b.w. per day. No adverse effects have been observed in the sub-chronic or long-term toxicity studies in mice or rats at the highest dose tested of 50000 mg CrO3 or chromium picolinate/kg diet. The relevant NOAELs corresponded to 506 and 286 mg Cr(III)/kg b.w. per day for the sub-chronic and long-term toxicity in the rat, respectively (NTP, 2010)”

 

Aluminium

In the study conducted by Katz et al. (1984) sodium aluminium phosphate (SALP) was administered to groups of six male and female beagle dogs at dietary concentrations of 0, 0.3, 1.0 or 3.0 % for 6 months. There were no statistically significant differences in mean body weights between test and control groups at any of the weekly determinations. Weekly mean food consumption values of all male treated groups did not differ significantly from those of the controls at any state of the study. Statistically significant reductions in food consumption occurred sporadically in all treated groups of female dogs. No significant absolute or relative organ-weight differences were found between any of the treated groups and their respective controls. Haematology, clinical chemistry and urinalysis data showed no toxicologically significant trends. Histopathology revealed no changes considered to be related to treatment. Thus, dietary administration of sodium aluminium phosphate for 6 months at concentrations of 3% or lower caused no significant toxicological effects in beagle dogs.

 

Additionally, the EFSA evaluation (2008, re-evaluation in 2018) concluded for the food additives aluminium sulphates (E 520–523) and sodium aluminium phosphate (E 541): “There is no concern with respect to genotoxicity and carcinogenicity. The no observed adverse effect level (NOAEL) for aluminium compounds in subchronic studies was 52 mg Al/kg body weight (bw) per day in rats and 90 mg Al/kg bw per day in dogs and the lowest NOAEL for neurotoxicity in rats was 30 mg Al/kg bw per day and for developing nervous system was 10–42 mg Al/kg bw per day in studies in mice and rats. The Panel concluded that aluminium sulphates (E 520–523) and sodium aluminium phosphate, acidic (E 541) are of no safety concern in the current authorised uses and use levels.”

 

Oral bioavailability of aluminium from aluminium compounds is low as a result of formation of insoluble aluminium hydroxide at the neutral pH in the intestines. Data from humans and experimental animals indicate that the bioavailability is approximately 0.3% of aluminium compounds in drinking water and 0.1% in food and beverages. Predicted plasma levels at the EFSA TWI of 1 mg Al/kg bw were within the reported normal levels of aluminium in serum, 1–3 lg/L. Absorbed aluminium accumulates in the bone. Kidney is the main route of excretion. Increased brain levels of aluminium have been reported in dialysis encephalopathy patients.

 

Iron

Considering the role of iron in human metabolic processes, it is highlighted that iron has several vital functions in the body. It serves as a carrier of oxygen to the tissues from the lungs by red blood cell haemoglobin, as a transport medium for electrons within cells, and as an integrated part of important enzyme systems in various tissues. It is an essential constituent of oxygen carriers, such as haemoglobin and myoglobin, and the iron contained within haem is essential for the redox reactions of numerous cytochromes. Insufficient intake results in the deficiency condition anaemia, adverse outcomes of pregnancy, impaired psychomotor development and cognitive performance and reduced immune function.

Any elemental iron in the diet is probably absorbed as non-haem iron following its dissolution in the acid stomach contents. The absorption of non-haem iron can be increased substantially by the presence of ligands, such as ascorbate, citrate and fumarate, as well as the presence of amino acids (e.g. cysteine) and oligopeptides resulting from meat digestion (Mulvihill et al., 1998). In contrast, very stable complexes, for example with phytates, phosphates and oxalates, impair non-haem iron absorption. Depending on the concentration of supportive or inhibitory ligands in the intestinal lumen the absorption of non-haem iron can vary by a factor of 10 in single-meal studies, but the effects are less pronounced in more long-term studies (Hallberg and Rossander, 1984; Rossander, 1987; Hunt and Roughead 2000). Iron is reversibly stored within the liver as ferritin and haemosiderin whereas it is transported between different compartments in the body by the protein transferrin. Iron excretion via the kidneys is very low, and body iron is highly conserved. Renal elimination is not controlled as part of iron homeostasis or the control of excess body stores. Normally, only about 0.1 mg is lost daily in urine. The sloughing of mucosal enterocytes results in elimination of absorbed iron before it reaches the systemic circulation and accounts for the loss of 0.6mg per day into the intestinal lumen. About 0.2-0.3 mg is lost daily from the skin. The total daily loss is equivalent to about 0.05 % of body iron content (Green et al., 1968).

 

Animal data

In a range finding study for carcinogenicity testing (Sat et al. 1985) ferric chloride hexahydrate was administered to Fischer 344 rats (10/sex/dose) via drinking water for 14 weeks, at concentrations of 0.12, 0.25, 0.5, 1.0 and 2.0 % FeCl3 (equivalent to approximately 80, 154, 277, 550 and 1231 mg/kg bw/day in male rats, 88, 176, 314, 571 and 1034 mg/kg bw/day in female rats). There were no deaths or clinical signs of toxicity. There was a significant reduction in body weight gains at the two highest doses at the end of the treatment period. A significant suppression of 17 % to 60 % in the intake of drinking water was observed in the groups given doses of 0.5 % and above. Treated males had increased levels of serum iron and higher red blood cell counts compared with controls. In microscopic examinations of sections stained with haematoxylin and eosin, brown pigment deposition was observed only in the keratin layers of the oesophageal mucosa in the groups given concentrations of 0.25 % or higher, and in the laminae propriae of the large intestine in the 2.0 % group. In sections stained with Berlin blue, increased numbers of positive pigments (an indication of iron overload) were also observed in the hepatocytes and Kupffer cells of the liver, the cartilage of the trachea and bronchus, the keratin mucosal layers of the tongue, the forestomach, the mucous layers of the small intestines, the white pulp of the spleen, the tubular epithelium of the kidney and the adipose tissues of the groups given 0.25% and higher. The intensity of the staining was marked in the intestine and liver. The NOAEL was 0.5 % (equivalent to 277 and 314 mg/kg bw/day in males and females, respectively) based on the reduced body weight gain.

 

Human data

The side effects of oral iron preparations increase with increase in dosage, but there are fewer side effects with slow delivery systems or if the iron is taken with food (Brock et al., 1985; Reddaiah et al., 1989). The adverse gastrointestinal effects are related to the concentration of iron in the intestinal lumen (Cook et al., 1990).

A daily dose of 50 mg of iron produced a higher incidence of gastrointestinal effects in subjects given conventional ferrous sulphate compared with subjects given the same amount in a wax-matrix (Brock et al., 1985), and also in subjects given ferrous sulphate compared with subjects given the same amount of iron as bis-glycino iron (Coplin et al., 1991). Neither of these studies included a placebo group, and therefore the association with iron is based on different responses to different preparations. A higher incidence of side effects was reported in subjects given 60 mg of iron as iron fumarate daily compared with placebo; daily doses of 120 mg of iron as fumarate for 8 weeks given to 19 young women in a double blind cross-over study resulted in gastrointestinal effects in 5 subjects while receiving iron, compared with 2 while taking placebo (Frykman et al., 1994).

Iron overload with clinical symptoms, which has only been found in adult subjects homozygous for hereditary haemochromatosis, those under long-term, high-dose medical treatment with iron, and those given repeated blood transfusions. Such information is of no relevance for the chemicals safety assessment.

 

(ii) in vitro bioaccessibility resultswithHematite chromium green black in several physiological media

For the pigmentHematite chromium green blackthis has been investigated experimentally in vitro by simulating dissolution under physiological conditions considered to mimic the most relevant exposure routes (oral, dermal and inhalation; Pardo Martinez, 2010). With a 100 mg loading at pH 1.5 (gastric fluid conditions) and an assumed gastric phase of digestion of about 2 hours, 7.8 μg/L of Al and <0.5 μg/L (<LOD) of Cr were dissolved. Based on 100 mg of pigment, this results in 0.01 wt-% dissolved Al. A relative bioaccessibility for chromium could not be determined, since the measured concentrations were below the limit of detection.

Sub-chronic inhalation toxicity

The absence of repeated dose toxicity via the inhalation route of Hematite chromium green black is deduced from (i) an absence of concern for the endpoint repeated dose toxicity via the inhalation route for the oxidic forms of the metals present in Hematite chromium green black, (ii) the low inhalability of hematite chromium green black by rats (based on MPPD modelling), (iii) the very clear finding that Hematite chromium green black is of poor bioavailability in a wide range of physiological media, (iv) Hematite chromium green black shows no effects in a number of in vitro and in vivo toxicological tests, and (v) the PSLT properties of Hematite chromium green black. Furthermore, under the umbrella of the MISA programme, a read-across for oral systemic and inhalation local effects is currently being developed for the inorganic pigments category. The read-across approach will consider all constituents of substances being a member of the inorganic pigments category. In the data matrix, representative members of the inorganic pigments category will be selected, based on the criteria (i) constituents of known toxicological activity present, (ii) highest relative bioavailability. Experimental studies will be conducted or proposed for these representative substances and read-across to other members of the inorganic pigments category.

Overall, the conduct of a 90-day repeated dose toxicity study via inhalation cannot be expected to contribute any relevant information to the assessment of (otherwise negative) information on repeated toxicity. As a result, the need for such testing is waived in accordance with regulation (EC) 1907/2008, Annex XI, Section 1.2. Based on the weight-of-evidence information for Hematite chromium green black as outlined below, it is concluded that Hematite chromium green black does not present a repeated dose toxicity hazard, as follows:

(i) Supporting information for repeated dose toxicity with metal oxides contained in Hematite chromium green black.

The elements chromium, aluminium and iron are present inhematite, chromium green black in oxidic form. It is therefore reasonable to read-across from the similarly poorly soluble/bioavailable oxides of these three metals to this pigment for hazard characterisation purposes, at the same time however considering the established very limited release of these three metals from the pigment matrix under physiological conditions.

Chromium(III) oxide

Derelanko et al. (1999) investigated the repeated dose inhalation toxicity of chromium oxide in groups of 15 male and 15 female CDF (Fischer 344/Crl BR VAF/Plus rats, which received the substance via nose-only inhalation at concentrations of 4.4, 15 and 44 mg/m³ air (actual concentration). No test item-related effects were observed for clinical signs, mortality, body weights, ophthalmological examination, haematology, clinical chemistry and urinalysis. In addition, no test item-related effects were found during a bronchoalveolar lavage evaluation.

A NOAEC of 15 mg Cr2O3/m³ was derived on the basis of a dose-dependent mild inflammatory response characterised by an increase of phagocytotic cells and a subsequent increase of lung weights being significant in the male animals of the high dose group. None of the BALF parameters (lactate dehydrogenase (LDH), total protein, beta-glucuronidase, and glutathione reductase) were affected after 90-day inhalation exposure, indicating a lack of tissue damage. No other adverse effects were observed, and the inflammatory response was completely reversible within the recovery period.

 

Aluminium oxide

The studies conducted by Gross et al. (1973) investigated the effects of chronic exposure (6 or 12 months) to different aluminium powders on the lungs of rat, hamsters and guinea pigs. Aluminium oxide dust was included as a negative “non-fibrogenic control”. A decrease in dead animals was observed in control as well as treated animals. Thus, this increase was not considered to be treatment related. Several endpoints specified in the 90-day inhalation toxicity guideline (OECD TG 413) were not assessed, particularly body and organ weights. It should be noted that the study was designed for the investigation of lung effects specifically. It is considered reliable for use in the hazard identification. Histopathology of lungs showed numerous relatively small foci centred around respiratory bronchioles and alveolar ducts. These foci were composed of clustered alveoli filled with well-preserved but swollen macrophages engorged with aluminium oxide. No adverse effects concentrations were observed in the sub-chronic toxicity studies in rat, hamster and guinea pigs at the highest dose tested of 70 mgAl2O3/m³ air.

 

Iron oxide

Wistar rats were nose-only exposed to pigment-sized iron oxide dust (Fe3O4, magnetite) in a sub chronic 13-week inhalation study according to the OECD testing guidelines TG#413 and GD#39 (Pauluhn, 2010). A 4-week pilot study with a 6 month post exposure period served as basis for validating the kinetic modelling approaches utilized to design the sub chronic study. Kinetic analyses made during this post exposure period demonstrated that a diminution in particle clearance and lung inflammation occurred at cumulative exposure levels exceeding the lung overload threshold.

The exposure to iron oxide dust was tolerated without mortality, consistent changes in body weights, food and water consumption or systemic toxicity. While general clinical pathology and urinalysis were unobtrusive, haematology revealed changes of unclear toxicological significance (minimally increased differential neutrophil counts in peripheral blood). Elevations of neutrophils in bronchoalveolar lavage (BAL) appeared to be the most sensitive endpoint of study. Histopathology demonstrated responses to particle deposition in the upper respiratory tract (goblet cell hyper- and/or metaplasia, intraepithelial eosinophilic globules in the nasal passages) and the lower respiratory tract (inflammatory changes in the bronchioalveolar region). Consistent changes suggestive of pulmonary inflammation were evidenced by BAL, histopathology, increased lung and lung-associated-lymph node (LALN) weights at 16.6 and 52.1 mg m−3. Increased septal collagenous fibres were observed at 52.1 mg/m3. Particle translocation into LALN occurred at exposure levels causing pulmonary inflammation. In summary, the retention kinetics iron oxide reflected that of poorly soluble particles. The empirical no-observed-adverse-effect level (NOAEL) and the lower bound 95% confidence limit on the benchmark concentration (BMCL) obtained by benchmark analysis was 4.7 and 4.4 mg/m3, respectively, and supports an OEL (time-adjusted chronic occupational exposure level) of 2 mg/m3 (alveolar fraction).

(ii) Inhalability assessment of hematite chromium green black by rats

When assessing the exposure of specific regions of the respiratory tract, the deposited material of the “head” and “tracheo-bronchial” region would actually impact in the nose, pharynx and bronchi and would subsequently predominantly be swallowed. Thus, the majority of the deposited material would contribute to oral and not to inhalation exposure. The deposited fraction in the “TB” region, however, is assumed to be discharged into the gastro-intestinal tract by mucociliary escalation and subsequent swallowing.

Consequently, a repeated dose inhalation toxicity study even under such worst-case conditions would result in an effective inhalation dose of approx. 1.55 mg/kg bw/day and an oral dose of approx. 98 mg/kg bw/day. Comparing the effective dose being available for absorption, one has to conclude that a repeated dose toxicity study via the oral route with a limit dose of 1000 mg/kg bw/day would result in a much higher dose being available for absorption. Such conclusion is however only valid for substance with no local effects upon inhalation.

 

(iii) PSLT property of the substance

The substance hematite, chromium green black is not expected to show any toxicity up to the limit dose of 1,000 mg/kg bw/d in a 28d oral toxicity test in rats based on read-across to the inorganic pigments manganese alumina pink corundum and chromium iron oxide. In an acute inhalation toxicity test no toxicity was observed up to the limit dose of 5mg/L. The solubility in artificial lung fluids (and also all other tested surrogate physiological media) has been shown to be well below 1 mg/L. Bioaccessibility data (Pardo Martinez, 2010) in Gamble’s solution at pH 7.4, which mimic the interstitial fluid within the deep lung under normal health conditions, revealed <0.5 μg/L of Cr (below L.O.D., 0.0009 wt-% based on the total Cr content) and 1.9±0.1 μg/L of Al (0.018 wt-% based on the total Al content). Likewise, in surrogate alveolar fluid conditions at pH 4.5 (which mimics the lysosomal environment after phagocytosis by macrophages),<0.5 μg/L of Cr (0.0009 wt-% based on the total Cr content) and 6.4±0.2 μg/L of Al (0.063 wt-% based on the total Al content) are dissolved. Based on 100 mg of pigment, this results in <0.0005/<0.0005 wt-% dissolved Cr and 0.0019/0.0064 wt-% dissolved Al.

 

Based on the lack of systemic and local toxicity as well the negligible oral bioavailability, the substance can be designated as being of low intrinsic toxicity. Due to its poor solubility in water and surrogate physiological media, it also qualifies as being of “poor solubility”. The German CA (BAuA, Dortmund) in their methodology paper (BAUA, 2017) on the identification of granular biopersistent particles in the workplace have defined a solubility cut-off < 1mg/L. The substance clearly meets this criterion and can therefore be identified as a candidate ”poorly soluble particle of low intrinsic toxicity” (“PSLT”).

 

Hence, the conduct of a sub chronic inhalation study cannot be expected to provide any relevant scientific information beyond what is already know about PSLT substances. In fact, the effects of inhalation exposure in laboratory animals for such PSLTs can safely be expected to be characterised by pulmonary overload associated with secondary inflammatory effects. This effect has been recently recognised by ECHA RAC (October 2017) in their opinion on the CLH proposal for titanium dioxide, designated as a mere physical, not substance-specific particle effect that is considered common to all PSLTs.

 

Such effects are already well established for the analogous pigment substance titanium dioxide. The overload effects have been characterised in rats, mice and hamsters (Bermudez et al., 2002), the rat being uniquely sensitive. Since both TiO and this substance equally qualify as PSLTs, the effects can be predicted as described for TiO2 in the study by Bermudez et al, namely the onset of inflammation at inhalation exposures>50 mg/m3due to macrophage overload and cessation of clearance. For animal welfare reasons, the repetition of such a study is not warranted, since the outcome can easily be anticipated not to yield any additional scientific information.

 

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.The no observed adverse effect level (NOAEL) in rats is 1000 mg/kg/day.

No classification for repeated dose toxicity according to EC Regulation No. 1272/2008 is anticipated.