Registration Dossier

Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

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

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
2008-10-03
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2014-05-14
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Manganese alumina pink corundum
EC Number:
269-061-0
EC Name:
Manganese alumina pink corundum
Cas Number:
68186-99-2
Molecular formula:
Mn(x)Al(2-x)O3 0,01≤x≤0,25
IUPAC Name:
Manganese alumina pink corundum
Test material form:
solid: particulate/powder
Details on test material:
- Chemical description: Manganese Alumina Pink Corundum
- Substance type: inorganic pigment
- Physical state: solid, pink powder, odourless, Hematite-corundum structure
- Storage condition of test material: at room temperature
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, kept dry and stored in a tightly closed container

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
Rats were selected because of their proven suitability in toxicology studies and to comply with regulatory requirements for testing in a rodent animal species.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at first dosing: males: 35 days; females: 36 days
- Weight at first dosing: males: 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

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The route of administration was selected according to the expected route of exposure.
Vehicle:
other: 0.8 % aqueous hydroxyl propyl methylcellulose gel
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in the vehicle to the appropriate concentration. The administration formulation was continuously agitated by stirring throughout the entire administration procedure.
The administration formulation was freshly prepared every day.
Administration volume: 10 mL/kg bw/day
The amount of the test item was adjusted to each animal's current body weight daily.

VEHICLE
- Source: FAGRON GmbH & Co. KG, 22885 Barsbüttel, Germany
- Batch 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
Doses / concentrations
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 males / 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: in agreement with the Sponsor and based on available toxicity data a limit test was performed.
Positive control:
none

Examinations

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

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

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

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

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

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

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

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

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

URINALYSIS: No

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

IMMUNOLOGY: No

TOXICOKINETC: Yes (please refer to Fraunhofer IME, report no. EBR-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)

Results and discussion

Results of examinations

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.

Effect levels

Key result
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
NOAEL (oral; rats) > 1000 mg 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