Cobalt zinc aluminate blue spinel

Endpoint summary

Description of key information

It is concluded that the pigment was well tolerated and that no signs of systemic toxicity whatsoever were observed had been seen in rats when administered at a dose of 1000 mg/kg bw/day for up to 28 days. Either no or only marginal increases in Al, Zn and Co plasma concentrations were observed, and only a minor fraction (<0.003%) of the total administered dose of Co, Al and Zn was collected via urine, documenting the lack of bioavailability of this pigment. The no observed adverse effect level (NOAEL) in rats is 1000 mg/kg/day.

 

In a 90 -day repeated dose toxicity by inhalation study, rats were exposed via nose-only inhalation towards aerosol concentrations of 0.4, 1.5 and 6 mg/m³ of cobalt zinc aluminate blue spinel. All animals survived the test period and were euthanized at scheduled dates. Effects indicating systemic toxicity were not observed. Sex-specific differences were not detected.The NOAEC was considered to be 6 mg/m³ (the concentration at or above the onset of lung overload, thus constituting the maximum tolerated concentration).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-01-16 to 2015-02-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-10-03

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2014-05-14

Limit test:

yes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, kept dry and stored in a tightly closed container

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at first dosing: males: 35 days; females: 36 days
- Weight at first dosing: males: 155.3 g - 178.7 g; females: 126.8 g - 148.4 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-149/6-27/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 the other study). In case of this pigment a hydrofluoric acid microwave assisted digestion (mixture of HNO3 and HF) 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. Die 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. To reduce the usage of the highly toxic hydrofluoric acid the digestion method was changed and tested in the beginning only at one application solution. 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 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. In the last step but only for the first application solution to the remaining solid material (only few white particles) a nitric acid/hydrofluoric acid solution was added and the samples were digested. After the evaluation of the data of the first application solution nearly no concentration of Al, Co and Zn could be found in this last step (see data evaluation/results). For this reason in the following application solution this last step was not performed to reduce the danger of using hydrofluoric acid.

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, 394.401 nm and 396.152 nm; cobalt was detected at the wavelength 228.615 nm, 230.786 nm, 231.160 nm and 231.406 nm and zinc was detected at the wavelength 202.548 nm, 206.200 nm and 213.857 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 0.999759. 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 and 396.152 nm
Co: 238.345 nm
Zn: 202.548 nm, 206.200 nm and 213.857 nm.

The applied LOD/LOQ calculations for the Agilent 720 ICP-OES are (according to DIN 32645) (Chemische Analytik - Nachweis-, Erfassungs- und Bestimmungsgrenze unter Wiederholbedingungen – Begriffe, Verfahren, Auswertung; German version DIN 32645:2008-11. Beuth Verlag.):
LOD: 3 * standard deviation of calibration blank/slope of the calibration
LOQ: 3 * LOD
The resulting LODs/LOQs are as follows:
- LOD: 0.344 µg/L (Al); 0.622 µg/L (Co); 0.141 µg/L (Zn)
- LOQ: 1.03 µg/L (Al); 1.87µg/L (Co); 0.423 µg/L (Zn)
- correlation coefficient: 0.999840 (Al); 0.999780 (Co); 0.999989 (Zn)
The certified reference materials 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 aluminium, cobalt and zinc (by standard addition of commercial standards) to determine the standard recovery of aluminium, cobalt and zinc. Data are compiled in Table 5 - Table 7. For fortified samples, recoveries were 98.9 - 115% for Al, 98.6 - 101% for Co and 99.0 - 110% for Zn.

Dose verification:
nominal dose: 1,000 mg/kg bw pigment (284 mg/kg bw Al, 153 mg/kg bw Co and 205 mg/kg bw Zn)
Results:
Analysis of stability and concentration (3 samples):
Recovery [%]:
Al. 92.5 - 102
Co: 88.6-99.6
Zn 87.7-96.6

Anaylsis of homogenity (3samples):
Recovery [%]:
Al: 96.6-98.7
Co: 92.6-94.9
Zn: 91.3 - 93.6

Anaylsis of concentration (1sample):
Recovery [%]:
Al: 94.9
Co: 91.1
Zn: 90.2

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-149/6-27/y)
Urine and plasma samples were obtained at study termination. Urine and plasma samples were analysed for aluminium, cobalt and zinc 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 or external appearance were noted for the male and female rats treated with 1000 mg cobalt zinc aluminate blue spinel/kg bw/day or for the animals treated with the vehicle control.
- all male and female rats treated with 1000 mg test item/kg bw/day revealed blue discoloured faeces as of test day 8 (not an adverse effect, since test item is a blue powder). The faeces of the control and test item-treated animals were formed normally.

MORTALITY
- no test item-related deaths occurred.
- 1/5 female control animals and 1/5 male animals of the test item-treated group 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 test item./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 in relative food consumption of test item-treated animals compared to the control animals were recorded (not test item-related findings):
males (test weeks 1 and 4): increased relative food consumption (p ≤ 0.05)
females (test weeks 2 and 4): decreased relative food consumption (p ≤ 0.01 or p ≤ 0.05).

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 cobalt zinc aluminate blue spinel/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 on haematological and coagulation parameters was noted for the male and female rats treated with 1000 mg cobalt zinc aluminate blue spinel/kg bw/day compared to the control group.
- statistically significant differences in haematological parameters of test item-treated animals compared to the control animals were recorded (not test item-related findings):
females (test day 29): increased platelets (control group: 806.2 ± 124.2 x 10³/µL vs. treatment group: 1090.4 ± 141.7 x 10³/µL; p ≤ 0.01) and decreased absolute basophilic granulocytes (control group: 0.022 ± 0.004 x 10³/µL vs. treatment group: 0.014 ± 0.005 x 10³/µL; p ≤ 0.05).
However, the stated haematological findings are within 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 (p ≤ 0.05) in biochemical parameters of test item-treated animals compared to the control animals were recorded (not test item-related findings):
males (test day 29): increased bilirubin (control group: 2.24 ± 0.15 µmol/L vs. treatment group: 2.54 ± 0.22 µmol/L; p ≤ 0.05)
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 cobalt zinc aluminate blue spinel/kg bw/day.
- examination results of the animals treated with the vehicle control were also in the normal range.

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 cobalt zinc aluminate blue spinel/kg bw/day compared to the control group.
- statistically significant differences (p ≤ 0.05) in organ weights of test item-treated animals compared with the control animals were recorded (not test item-related findings):
males (test day 29): decreased relative spleen weight (control group: 2.245 ± 0.230 g/kg bw vs. treatment group: 1.910 ± 0.159 g/kg bw; p ≤ 0.05) and decreased absolute spleen weight (control group: 0.692 ± 0.076 g vs. treatment group: 0.572 ± 0.059 g/kg bw; p ≤ 0.05).
However, the relative and absolute spleen weight are within the normal range for that rat strain and 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 test item/kg bw/day revealed any test item-related macroscopic changes at necropsy on test day 29.
- 3/5 male and 4/5 female animals treated with 1000 mg test item/kg bw/day revealed a green discoloured content of the stomach or the intestines (colon, ileum, jejunum and/or rectum)(findings considered to be due to the test item; not an adverse effect).

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 group and the treatment group).
- granular to filamentary green-coloured material was noted in the intestine lumen of the male and female rats of treatment group. The epithelial cells of the small and large intestine were normal without any inflammatory or degenerative reactions. This finding correlated with the green discoloured content of the intestines noted at necropsy .

- inflammatory lesions occurred in various organs such as liver, trachea, larynx, kidney, and epididymis 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. No difference between control group and treatment group.

- 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 the treatment group.

- a minimal to mild single cell or peripheral fatty infiltrations in the hepatocytes of the liver and a minimal to mild 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 and the treatment group. Fatty infiltration in the hepatocytes of the liver 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.

- a minimal 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 groups and the involution of the thymus in the rats of both groups corresponded in type, incidence and severity to the age of the animals.

- Coincidental findings in a small number of control- and test animals are:
Kidney: basophilic tubular cells;
Larynx: glandular ectasia;
Mammary glands: glandular hyperplasia particularly in male rat;
Ovary: follicular cysts, corpus luteum cysts;
Stomach: glandular ectasia;
Testis: atrophy of the germinative epithelium;
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 treatment group in male and female rats.
Please also refer to the field "Attached background material" below.

TOXICOKINETICS
Cobalt, aluminium and zinc are of negligible bioavailability from the test substance Cobalt zinc aluminate blue spinel: by recalculating the urine levels and setting them into relation to the administered dose of the individual elements Co, Al and Zn, 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 all three 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 cobalt zinc aluminate blue spinel/kg bw/day

No test item-related changes were observed for clinical signs, mortality, neurologically screening, body weight, food consumption, water consumption, haematology, clinical chemistry, organ weights, ophthalmology, gross pathology, and histopathology.
The uptake of cobalt, aluminium and zinc 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 three metals, mirrored by either minimal or no increases in blood plasma concentrations. This supports the assumption that all three elements are not biologically available upon ingestion of the pigment Cobalt zinc aluminate blue spinel.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-02-25 to 2021-09-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study

Version / remarks:

2018-06-25

Deviations:

yes

Remarks:

Ophthalmology not performed (this endpoint is not sensitive in particle studies); urine analysis not performed (endpoint optional in guideline)

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate signed 2018-11-22

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, dry, protected from light.

Species:

rat

Strain:

Wistar

Remarks:

Wistar (Han)

Details on species / strain selection:

Wistar rats are commonly used in subchronic and chronic inhalation toxicity studies. They fulfil the criteria stated by a U.S. EPA Workshop (Vu et al., 1996)* such as (i) a low background rate of neoplasia, (ii) a low background rate of pulmonary disease, (iii) longevity, and (iv) a history of laboratory use.

*References:
- Vu, V., Barrett, J.C, Roycroft, J., Schuman, L., Dankovic, D., 1996. Workshop report: Chronic inhalation toxicity and carcinogenicity testing of respirable fibrous particles. Reg Tox Pharm 24, 202-212.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld, Germany)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: approx. 250 g for males and approx. 175 g for females
- Housing: housed in Makrolon (polycarbonate) cages type III with softwood (‘ssniff BK 8-15’) bedding material.
- Diet: commercial chow in pellet form (ssniff “V1534”) purchased from ssniff Spezialdiäten GmbH (Soest, Germany); ad libitum
- Water: tap water; ad libitum
- Acclimation period: Approx. one week the animals will be allowed to adjust and become acclimatised to the Fraunhofer ITEM environment. During the 2-3 weeks prior exposure start, all rats will be trained to the 6-hour restraint in nose-only tubes.

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

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1.26 - <= 1.57 µm

Remarks on MMAD:

MMAD / GSD: please refer to Table 1 ('Any other information on materials and methods incl. tables')

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow-past nose-only inhalation exposure system
- Method of holding animals in test chamber: restrain tubes with a flexible stopper
- System of generating particulates/aerosols: The particulate sample aerosols were generated by dry dispersion with pressurized air. Cyclones (in line) were used to reduce the coarse moiety of the aerosol. For each nose-only exposure unit, the aerosol was generated by a high-pressure pneumatic disperser. The disperser was fed with the test/reference items under computerized control, i.e. with a feed back loop to the actual aerosol concentrations measured by an aerosol photometer. The photometer gives a scattering light signal which is proportional to the particle concentration, if the particle size distribution is constant. The ratio between photometer signal and concentration was determined throughout the study by comparing to gravimetric concentrations.
- Temperature, humidity, pressure in air chamber: Parameters were recorded by 20-minute means The were set at 22°C + 2°C for temperature and 55% + 15% for relative humidity.
- Air flow rate: 1 L/min
- Method of particle size determination: The MMAD was determined four times (once before exposure start and once per month during the exposure period for each test item exposure unit (3 units) by a cascade impactor (Marple impactor).
- Treatment of exhaust air: exhaled air is drawn off immediately by a cylinder surrounding the aerosol delivery cylinder

TEST ATMOSPHERE
- Brief description of analytical method used: Filter samples of the aerosols were taken daily to control the aerosol concentrations and to calibrate the aerosol photometers. The means are close to the target concentrations. Permanent control of the aerosol concentrations is guaranteed by photometers
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- see above ("Details on inhalation exposure")
- The target aerosol concentrations of 0.4, 1.5 and 6 mg/m³ Cobalt Zinc Aluminate Blue Spinel were achieved exactly, i.e. to 100% in each group.

Duration of treatment / exposure:

13 weeks (65 exposure days)

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

0.4 mg/m³ air (analytical)

Remarks:

SD: ± 0.02 mg/m³

Dose / conc.:

1.5 mg/m³ air (analytical)

Remarks:

SD: ± 0.05 mg/m³

Dose / conc.:

6 mg/m³ air (analytical)

Remarks:

SD: ± 0.23 mg/m³

No. of animals per sex per dose:

10+5 males and 10 females (1 day recovery); 5 males (28 days recovery); 5 males and females (90 days recovery) (total: 100 males and 60 females)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The concentrations were defined based on the preceding intratracheal instillation dose range finding (DRF A) study (Fraunhofer ITEM no. 02 N 20 502).
- Post-exposure recovery period: 1, 28, and 90 days

For the nominal aerosol concentrations of 0.4, 1.5 and 6 mg/m³ the test item deposition in the respiratory tract was modeled using the MPPD model (version 3.04), resulting in a deposited fraction of 4.6% (rel. density=4.5, MMAD/GSD=1.8µm/1.5).
This deposited fraction was used to calculate the total deposited mass, using the following input parameters:
Morphometry: Semi-symmetric Long Evans
Example for deposited mass at 0.4 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 0.4 mg/m³ x 4.7% = 0.03 mg/lung
Example for deposited mass at 1.5 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 1.5 mg/m³ x 4.7% = 0.125 mg/lung
Example for deposited mass at 6 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 6 mg/m³ x 4.7% = 1.0 mg/lung

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day (with the exception of weekends and public holidays: once daily)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before sacrifice

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

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

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

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

OPHTHALMOSCOPIC EXAMINATION: No

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

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

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

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

LUNG BURDEN: Yes
- Time schedule for analysis: 1, 28, and 90 after the 90-day exposure period
- Dose groups that were examined: all
- Number of animals: 5 male rats
- Chemical analysis: After sacrifice the lungs were prepared by freeze drying (> 6 hours (0.37 mbar), plasma ashing (> 24 hours, cool plasma conditions), and microwave (wet) digestion (H2SO4 (96%); max. 500 W). The test items retained in lung tissue were determined using ion-coupled plasma mass spectroscopy (ICP-MS) using the prepared samples after recommended dilution with deionized water.
Please refer to IUCLID section 7.1.1 "w_Creutzenberg_2022_lung burden" for more details on the method of lung burden analysis.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- rats were sacrificed 1, 28, and 90 days after the 90-day exposure period
- macroscopic examination: all animals were subjected to a complete necropsy
- organ weights were determined for the following organs: liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, thymus, spleen, brain, lung, and heart
HISTOPATHOLOGY: Yes
- In the study the organs according to OECD guideline 413 were examined of the female and male animals of the clean air control and pigment 1 (Cobalt Zinc Aluminate Blue Spinel) high dose group after 90 days nose-only inhalation: adrenals, aorta, bone marrow (and/or fresh aspirate), brain (including sections of cerebrum, cerebellum, and medulla/pons), caecum, coagulating glands, colon, duodenum, epididymides, femur and stifle joint, heart, ileum, jejunum, kidneys, larynx (3 levels incl. the base of the epiglottis), liver, lung (left lobe at three levels, including main bronchi), lymph nodes from the hilar region of the lung, lymph nodes (distal from the portal-of-entry), mammary gland (males and females), muscle (thigh), nasopharyngeal tissues (at least 4 levels; 1 level to include the nasopharyngeal duct and the Nasal Associated Lymphoid Tissue (NALT)), oesophagus, olfactory bulb, ovaries, pancreas, parathyroids, peripheral nerve (sciatic or tibial), pituitary, prostate, rectum, salivary glands, seminal vesicles, skin, spinal cord (cervical- mid-thoracic, and lumbar), spleen, sternum, stomach, teeth, testes, thymus, thyroid, trachea (at least two levels incl. 1 longitudinal section through the carina and 1 transverse section), urinary bladder, uterus, vagina, all gross lesions and masses.
The following respiratory tract organs of animals 101-110 and 201-210 of group 1 and 4, respectively, were examined histopathologically: Nasal and Paranasal Cavities, pharynx (Laryngopharynx/Nasopharynx), larynx, trachea, lung, lung-associated lymph nodes (LALN).
- All organs were preserved and fixed in formalin at day 1 and 90 post-exposure. Histopathology will be performed in 10 animals per sex of the control and high dose group at day 1 post-exposure.

Statistics:

Differences between groups will be considered statistically significant at p < 0.05. Data will be analysed using analysis of variance. If the group means differ significantly by the analysis of variance, the means of the treated groups will be compared with the means of the control groups using Dunnett’s test. The statistical evaluation of the histopathological findings will be done with the two-tailed Fisher test by the PROVANTIS system.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Lungs showed typical dose-dependent discolourations caused by the test item:
- 1 day post exposure: 1/20 males and 1/10 females of the control group, 3/20 males and 1/10 females of the low dose group, 5/20 males and 4/10 females of the mid dose group, and 18/20 males and 10/10 females of the high dose group
- 90 days post exposure: 1/5 males and 0/5 females of the controls, 0/5 males and 3/5 females of the low dose group, 2/5 males and 1/5 females of the mid dose group, and 4/5 males and 5/5 females of the high dose group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

High dose group:
- In the lung, exposure-related findings represent very slight multifocal granulocytic cell infiltration in the alveoli in 6/10 male and 5/10 female rats. Particle-laden macrophages were observed multifocally in the alveoli in 10/10 males (7 slight; 3 moderate) and 10/10 females (8 slight; 2 moderate) as well as in the bronchus-associated lymphoid tissue in 6/10 males (all very slight) and in 7/10 females (all very slight). This lesion correlated with a macroscopically observed multifocal discoloration of up to 1 mm in diameter in the lung. A multifocal very slight bronchiolo-alveolar hyperplasia of the bronchial type (bronchiolization) was seen in 1/10 males.
- In the nasal cavity, a very slight multifocal accumulation of particle-laden macrophages was found in 3/10 males and 3/10 females in the nose-associated lymphoid tissue (NALT). Further, very slight multifocal hyaline droplets were seen in the nasal epithelium in 1/10 male and 2/10 female rats.
- In the lung-associated lymph nodes (LALN), there was a multifocal accumulation of particle-laden macrophages in 10/10 males (7 very slight; 3 slight) and in 7/10 (3 very slight; 4 slight) females. This lesion correlated with a macroscopically observed enlargement in few animals.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

ORGAN WEIGHT FINDINGS INCL ORGAN / BODY WEIGHT RATIOS:
- At day-1 after the last exposure, females of the low and high dose groups showed statistically significantly increased absolute (+26.3% and +29.9%, respectively) and relative (+23.4% and +26.8%) weights of the left adrenal, when compared to the vehicle control group. However, no such effects were observed for the right adrenals. Moreover, the effect was transient, no effects on adrenal weights were observed in any male dose group, and the finding in females was without a histopathological correlate. Thus, the findings are considered to be not biologically relevant.
- At day-1 after the last exposure, females of the high dose group showed marginally but statistically significantly increased absolute and relative weights of the left kidney (+10.5% and 8.4%, respectively), when compared to the vehicle control group. However, no such effects were observed for the right kidneys. Moreover, the effect was transient, no effects on kidney weights were observed in any male dose group, and the finding in females was without a histopathological correlate. Thus, the findings are considered to be not biologically relevant.

GROSS PATHOLOGICAL FINDINGS:
- Enlarged lung-associated lymph nodes (LALN), as a particle-specific lung clearance pathway, were observed only incidentally (1 day post exposure: 2/20 males and 1/10 females of the high dose group; 90 days post exposure: 1/5 females of the control and low dose group).
- Some other incidental macroscopic observations in other organs were obtained (1 day post exposure: 1/20 males of the low dose group, 1/20 males and 1/10 females of the mid dose group, and 2/20 males and 1/10 females of the high dose group; 90 days post exposure: 1/5 females of the control group, 1/5 females of the mid dose group, and 1/5 females of the high dose group).

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC:
- All the other findings within the different organs occurred in single animals and were interpreted to be incidental without any relation to the exposure.

HAEMATOLOGICAL FINDINGS:
- On day 1 post exposure, the male low dose group showed a statistically significant decrease of the red blood cell count (-4.4%), when compared to the vehicle control group. Moreover, the haemoglobin concentration was statistically significantly decrease in the mid dose group (-3.1%). The haematocrit value was statistically significantly decreased in the low and mid dose group (-3.6% and -3.7%, respectively). Furthermore, the differential cell count revealed a statistically significant increase (+46.2%) in the proportion of banded neutrophils in the male low dose group, when compared to the vehicle control group. These effects were marginal, not dose-related, and not observed in females. Thus, the findings are considered to be not biologically relevant.

BALF ANALYSES:
- At days 1 and 90 (females only) post-exposure no statistically significant increases of polymorphonuclear neutrophils were detected in any treatment group. The PMN percentages (in the range from 1.2% to 3.3%, males - 1.3% to 2.0%, females) are close to historical clean air control data; thus, the test item did not induce a relevant PMN-related lung inflammation. Lymphocyte levels were also found low and at control levels (<1%).
- For lactic dehydrogenase, ß-glucuronidase and total protein, no relevant statistically significant increases were detected in any group of both sexes and sacrifice dates.

LUNG BURDEN ANALYSIS
- Lung weight: The lung burden analysis (males only) did not show any statistically significantly increase in the lung to bodyweight ratio at post-exposure day-1, 28, and 92, when compared to the vehicle control group.
- Chemical analysis: Information will be added to the robust study summary upon availability
Please refer to IUCLID section 7.1.1 "w_Creutzenberg_2022_lung burden" for more details on results of the lung burden analysis.

Key result

Dose descriptor:

NOAEC

Effect level:

6 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: observed at lung overload conditions, in exceedance of the maximum tolerated dose (MTD)

Critical effects observed:

no

Conclusions:

In this 90-day repeated dose toxicity by inhalation study, rats were exposed via nose-only inhalation towards aerosol concentrations of 0.4, 1.5 and 6 mg/m³ of Cobalt Zinc Aluminate Blue Spinel.

All animals survived the test period and were euthanized at scheduled dates. Effects indicating systemic toxicity were not observed. Sex-specific differences were not detected.
No relevant statistically significant changes as compared to concurrent controls were observed for: body weight and body weight development, food and water consumption, haematology, clinical chemistry, lung weights, bronchoalveolar lavage fluid (BALF) analysis, histopathological evaluation.
Some adaptive exposure-related findings were detected in the investigated high dose group (Pigment 1 - Cobalt Zinc Aluminate Blue Spinel) within the lung, the nasal cavity, and the lung-associated lymph nodes: accumulation of particle-laden macrophages. Very slight multifocal hyaline droplets were seen in the nasal epithelium. These lesions are considered to represent a non-adverse adaptive change. Moreover, very slight multifocal granulocytic cell infiltration in the alveoli were observed. A single male showed very slight bronchiolo-alveolar hyperplasia of the bronchial type (bronchiolization).

Under the conditions of this test, based on very slight granulocytic cell infiltration in the lung, a NOAEC of 6 mg/m³ was derived for both sexes, being the maximum tolerated concentration based on the more than 2-fold increase lung clearance half-time (further details are reported in the study record in the toxicokinetics section). Consequently, the pigment is to be regarded as inert material when tested up to the maximum tolerated concentration as defined by significant impairment of lung clearance (Driscoll and Borm, 2020)*.

*References:
- Driscoll, K.E. and Borm, P.J.A., 2020. Expert workshop on the hazards and risks of poorly soluble low toxicity particles. Inhal Toxicol 32(2):53-62.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-02-25 to 2021-09-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study

Version / remarks:

2018-06-25

Deviations:

yes

Remarks:

Ophthalmology not performed (this endpoint is not sensitive in particle studies); urine analysis not performed (endpoint optional in guideline)

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate signed 2018-11-22

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, dry, protected from light.

Species:

rat

Strain:

Wistar

Remarks:

Wistar (Han)

Details on species / strain selection:

Wistar rats are commonly used in subchronic and chronic inhalation toxicity studies. They fulfil the criteria stated by a U.S. EPA Workshop (Vu et al., 1996)* such as (i) a low background rate of neoplasia, (ii) a low background rate of pulmonary disease, (iii) longevity, and (iv) a history of laboratory use.

*References:
- Vu, V., Barrett, J.C, Roycroft, J., Schuman, L., Dankovic, D., 1996. Workshop report: Chronic inhalation toxicity and carcinogenicity testing of respirable fibrous particles. Reg Tox Pharm 24, 202-212.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld, Germany)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: approx. 250 g for males and approx. 175 g for females
- Housing: housed in Makrolon (polycarbonate) cages type III with softwood (‘ssniff BK 8-15’) bedding material.
- Diet: commercial chow in pellet form (ssniff “V1534”) purchased from ssniff Spezialdiäten GmbH (Soest, Germany); ad libitum
- Water: tap water; ad libitum
- Acclimation period: Approx. one week the animals will be allowed to adjust and become acclimatised to the Fraunhofer ITEM environment. During the 2-3 weeks prior exposure start, all rats will be trained to the 6-hour restraint in nose-only tubes.

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

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1.26 - <= 1.57 µm

Remarks on MMAD:

MMAD / GSD: please refer to Table 1 ('Any other information on materials and methods incl. tables')

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow-past nose-only inhalation exposure system
- Method of holding animals in test chamber: restrain tubes with a flexible stopper
- System of generating particulates/aerosols: The particulate sample aerosols were generated by dry dispersion with pressurized air. Cyclones (in line) were used to reduce the coarse moiety of the aerosol. For each nose-only exposure unit, the aerosol was generated by a high-pressure pneumatic disperser. The disperser was fed with the test/reference items under computerized control, i.e. with a feed back loop to the actual aerosol concentrations measured by an aerosol photometer. The photometer gives a scattering light signal which is proportional to the particle concentration, if the particle size distribution is constant. The ratio between photometer signal and concentration was determined throughout the study by comparing to gravimetric concentrations.
- Temperature, humidity, pressure in air chamber: Parameters were recorded by 20-minute means The were set at 22°C + 2°C for temperature and 55% + 15% for relative humidity.
- Air flow rate: 1 L/min
- Method of particle size determination: The MMAD was determined four times (once before exposure start and once per month during the exposure period for each test item exposure unit (3 units) by a cascade impactor (Marple impactor).
- Treatment of exhaust air: exhaled air is drawn off immediately by a cylinder surrounding the aerosol delivery cylinder

TEST ATMOSPHERE
- Brief description of analytical method used: Filter samples of the aerosols were taken daily to control the aerosol concentrations and to calibrate the aerosol photometers. The means are close to the target concentrations. Permanent control of the aerosol concentrations is guaranteed by photometers
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- see above ("Details on inhalation exposure")
- The target aerosol concentrations of 0.4, 1.5 and 6 mg/m³ Cobalt Zinc Aluminate Blue Spinel were achieved exactly, i.e. to 100% in each group.

Duration of treatment / exposure:

13 weeks (65 exposure days)

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

0.4 mg/m³ air (analytical)

Remarks:

SD: ± 0.02 mg/m³

Dose / conc.:

1.5 mg/m³ air (analytical)

Remarks:

SD: ± 0.05 mg/m³

Dose / conc.:

6 mg/m³ air (analytical)

Remarks:

SD: ± 0.23 mg/m³

No. of animals per sex per dose:

10+5 males and 10 females (1 day recovery); 5 males (28 days recovery); 5 males and females (90 days recovery) (total: 100 males and 60 females)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The concentrations were defined based on the preceding intratracheal instillation dose range finding (DRF A) study (Fraunhofer ITEM no. 02 N 20 502).
- Post-exposure recovery period: 1, 28, and 90 days

For the nominal aerosol concentrations of 0.4, 1.5 and 6 mg/m³ the test item deposition in the respiratory tract was modeled using the MPPD model (version 3.04), resulting in a deposited fraction of 4.6% (rel. density=4.5, MMAD/GSD=1.8µm/1.5).
This deposited fraction was used to calculate the total deposited mass, using the following input parameters:
Morphometry: Semi-symmetric Long Evans
Example for deposited mass at 0.4 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 0.4 mg/m³ x 4.7% = 0.03 mg/lung
Example for deposited mass at 1.5 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 1.5 mg/m³ x 4.7% = 0.125 mg/lung
Example for deposited mass at 6 mg/m³: 0.2 l minute breathing volume x 360 min exposure/day x 65 exposure days x 6 mg/m³ x 4.7% = 1.0 mg/lung

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day (with the exception of weekends and public holidays: once daily)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before sacrifice

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

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

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

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

OPHTHALMOSCOPIC EXAMINATION: No

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

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

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

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

LUNG BURDEN: Yes
- Time schedule for analysis: 1, 28, and 90 after the 90-day exposure period
- Dose groups that were examined: all
- Number of animals: 5 male rats
- Chemical analysis: After sacrifice the lungs were prepared by freeze drying (> 6 hours (0.37 mbar), plasma ashing (> 24 hours, cool plasma conditions), and microwave (wet) digestion (H2SO4 (96%); max. 500 W). The test items retained in lung tissue were determined using ion-coupled plasma mass spectroscopy (ICP-MS) using the prepared samples after recommended dilution with deionized water.
Please refer to IUCLID section 7.1.1 "w_Creutzenberg_2022_lung burden" for more details on the method of lung burden analysis.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- rats were sacrificed 1, 28, and 90 days after the 90-day exposure period
- macroscopic examination: all animals were subjected to a complete necropsy
- organ weights were determined for the following organs: liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, thymus, spleen, brain, lung, and heart
HISTOPATHOLOGY: Yes
- In the study the organs according to OECD guideline 413 were examined of the female and male animals of the clean air control and pigment 1 (Cobalt Zinc Aluminate Blue Spinel) high dose group after 90 days nose-only inhalation: adrenals, aorta, bone marrow (and/or fresh aspirate), brain (including sections of cerebrum, cerebellum, and medulla/pons), caecum, coagulating glands, colon, duodenum, epididymides, femur and stifle joint, heart, ileum, jejunum, kidneys, larynx (3 levels incl. the base of the epiglottis), liver, lung (left lobe at three levels, including main bronchi), lymph nodes from the hilar region of the lung, lymph nodes (distal from the portal-of-entry), mammary gland (males and females), muscle (thigh), nasopharyngeal tissues (at least 4 levels; 1 level to include the nasopharyngeal duct and the Nasal Associated Lymphoid Tissue (NALT)), oesophagus, olfactory bulb, ovaries, pancreas, parathyroids, peripheral nerve (sciatic or tibial), pituitary, prostate, rectum, salivary glands, seminal vesicles, skin, spinal cord (cervical- mid-thoracic, and lumbar), spleen, sternum, stomach, teeth, testes, thymus, thyroid, trachea (at least two levels incl. 1 longitudinal section through the carina and 1 transverse section), urinary bladder, uterus, vagina, all gross lesions and masses.
The following respiratory tract organs of animals 101-110 and 201-210 of group 1 and 4, respectively, were examined histopathologically: Nasal and Paranasal Cavities, pharynx (Laryngopharynx/Nasopharynx), larynx, trachea, lung, lung-associated lymph nodes (LALN).
- All organs were preserved and fixed in formalin at day 1 and 90 post-exposure. Histopathology will be performed in 10 animals per sex of the control and high dose group at day 1 post-exposure.

Statistics:

Differences between groups will be considered statistically significant at p < 0.05. Data will be analysed using analysis of variance. If the group means differ significantly by the analysis of variance, the means of the treated groups will be compared with the means of the control groups using Dunnett’s test. The statistical evaluation of the histopathological findings will be done with the two-tailed Fisher test by the PROVANTIS system.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Lungs showed typical dose-dependent discolourations caused by the test item:
- 1 day post exposure: 1/20 males and 1/10 females of the control group, 3/20 males and 1/10 females of the low dose group, 5/20 males and 4/10 females of the mid dose group, and 18/20 males and 10/10 females of the high dose group
- 90 days post exposure: 1/5 males and 0/5 females of the controls, 0/5 males and 3/5 females of the low dose group, 2/5 males and 1/5 females of the mid dose group, and 4/5 males and 5/5 females of the high dose group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

High dose group:
- In the lung, exposure-related findings represent very slight multifocal granulocytic cell infiltration in the alveoli in 6/10 male and 5/10 female rats. Particle-laden macrophages were observed multifocally in the alveoli in 10/10 males (7 slight; 3 moderate) and 10/10 females (8 slight; 2 moderate) as well as in the bronchus-associated lymphoid tissue in 6/10 males (all very slight) and in 7/10 females (all very slight). This lesion correlated with a macroscopically observed multifocal discoloration of up to 1 mm in diameter in the lung. A multifocal very slight bronchiolo-alveolar hyperplasia of the bronchial type (bronchiolization) was seen in 1/10 males.
- In the nasal cavity, a very slight multifocal accumulation of particle-laden macrophages was found in 3/10 males and 3/10 females in the nose-associated lymphoid tissue (NALT). Further, very slight multifocal hyaline droplets were seen in the nasal epithelium in 1/10 male and 2/10 female rats.
- In the lung-associated lymph nodes (LALN), there was a multifocal accumulation of particle-laden macrophages in 10/10 males (7 very slight; 3 slight) and in 7/10 (3 very slight; 4 slight) females. This lesion correlated with a macroscopically observed enlargement in few animals.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

ORGAN WEIGHT FINDINGS INCL ORGAN / BODY WEIGHT RATIOS:
- At day-1 after the last exposure, females of the low and high dose groups showed statistically significantly increased absolute (+26.3% and +29.9%, respectively) and relative (+23.4% and +26.8%) weights of the left adrenal, when compared to the vehicle control group. However, no such effects were observed for the right adrenals. Moreover, the effect was transient, no effects on adrenal weights were observed in any male dose group, and the finding in females was without a histopathological correlate. Thus, the findings are considered to be not biologically relevant.
- At day-1 after the last exposure, females of the high dose group showed marginally but statistically significantly increased absolute and relative weights of the left kidney (+10.5% and 8.4%, respectively), when compared to the vehicle control group. However, no such effects were observed for the right kidneys. Moreover, the effect was transient, no effects on kidney weights were observed in any male dose group, and the finding in females was without a histopathological correlate. Thus, the findings are considered to be not biologically relevant.

GROSS PATHOLOGICAL FINDINGS:
- Enlarged lung-associated lymph nodes (LALN), as a particle-specific lung clearance pathway, were observed only incidentally (1 day post exposure: 2/20 males and 1/10 females of the high dose group; 90 days post exposure: 1/5 females of the control and low dose group).
- Some other incidental macroscopic observations in other organs were obtained (1 day post exposure: 1/20 males of the low dose group, 1/20 males and 1/10 females of the mid dose group, and 2/20 males and 1/10 females of the high dose group; 90 days post exposure: 1/5 females of the control group, 1/5 females of the mid dose group, and 1/5 females of the high dose group).

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC:
- All the other findings within the different organs occurred in single animals and were interpreted to be incidental without any relation to the exposure.

HAEMATOLOGICAL FINDINGS:
- On day 1 post exposure, the male low dose group showed a statistically significant decrease of the red blood cell count (-4.4%), when compared to the vehicle control group. Moreover, the haemoglobin concentration was statistically significantly decrease in the mid dose group (-3.1%). The haematocrit value was statistically significantly decreased in the low and mid dose group (-3.6% and -3.7%, respectively). Furthermore, the differential cell count revealed a statistically significant increase (+46.2%) in the proportion of banded neutrophils in the male low dose group, when compared to the vehicle control group. These effects were marginal, not dose-related, and not observed in females. Thus, the findings are considered to be not biologically relevant.

BALF ANALYSES:
- At days 1 and 90 (females only) post-exposure no statistically significant increases of polymorphonuclear neutrophils were detected in any treatment group. The PMN percentages (in the range from 1.2% to 3.3%, males - 1.3% to 2.0%, females) are close to historical clean air control data; thus, the test item did not induce a relevant PMN-related lung inflammation. Lymphocyte levels were also found low and at control levels (<1%).
- For lactic dehydrogenase, ß-glucuronidase and total protein, no relevant statistically significant increases were detected in any group of both sexes and sacrifice dates.

LUNG BURDEN ANALYSIS
- Lung weight: The lung burden analysis (males only) did not show any statistically significantly increase in the lung to bodyweight ratio at post-exposure day-1, 28, and 92, when compared to the vehicle control group.
- Chemical analysis: Information will be added to the robust study summary upon availability
Please refer to IUCLID section 7.1.1 "w_Creutzenberg_2022_lung burden" for more details on results of the lung burden analysis.

Key result

Dose descriptor:

NOAEC

Effect level:

6 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: observed at lung overload conditions, in exceedance of the maximum tolerated dose (MTD)

Critical effects observed:

no

Conclusions:

In this 90-day repeated dose toxicity by inhalation study, rats were exposed via nose-only inhalation towards aerosol concentrations of 0.4, 1.5 and 6 mg/m³ of Cobalt Zinc Aluminate Blue Spinel.

All animals survived the test period and were euthanized at scheduled dates. Effects indicating systemic toxicity were not observed. Sex-specific differences were not detected.
No relevant statistically significant changes as compared to concurrent controls were observed for: body weight and body weight development, food and water consumption, haematology, clinical chemistry, lung weights, bronchoalveolar lavage fluid (BALF) analysis, histopathological evaluation.
Some adaptive exposure-related findings were detected in the investigated high dose group (Pigment 1 - Cobalt Zinc Aluminate Blue Spinel) within the lung, the nasal cavity, and the lung-associated lymph nodes: accumulation of particle-laden macrophages. Very slight multifocal hyaline droplets were seen in the nasal epithelium. These lesions are considered to represent a non-adverse adaptive change. Moreover, very slight multifocal granulocytic cell infiltration in the alveoli were observed. A single male showed very slight bronchiolo-alveolar hyperplasia of the bronchial type (bronchiolization).

Under the conditions of this test, based on very slight granulocytic cell infiltration in the lung, a NOAEC of 6 mg/m³ was derived for both sexes, being the maximum tolerated concentration based on the more than 2-fold increase lung clearance half-time (further details are reported in the study record in the toxicokinetics section). Consequently, the pigment is to be regarded as inert material when tested up to the maximum tolerated concentration as defined by significant impairment of lung clearance (Driscoll and Borm, 2020)*.

*References:
- Driscoll, K.E. and Borm, P.J.A., 2020. Expert workshop on the hazards and risks of poorly soluble low toxicity particles. Inhal Toxicol 32(2):53-62.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Animal data - oral route:

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

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

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

 

Furthermore, individual 24-hour urine samples were collected from all animals after the last dosing prior to the scheduled sacrifice, and in addition plasma samples were collected from each animal at the day of sacrifice. The plasma and urine samples were analysed for total cobalt, zinc and aluminium content. The comparison of the total administered final pigment dose of 1000mg/kg bw with the total mean Co, Zn and Al content recovered in 24-urine samples, as calculated from the mean 24h-urine collection volumes of 11.6 ml for males and of 8.8 ml for females, would correspond to a total bioavailable cobalt fraction of 0.0026% for males and 0.0027% for females, a total bioavailable zinc fraction of 0% for males and females, and a total bioavailable aluminium fraction of 0% for males and females (under the simplified assumption that excretion of these three elements occurs to a relevant extent via urine which is the case for aluminium and cobalt, whereas for zinc pancreatic secretion also plays a role). The Co, Zn and Al concentrations of plasma samples, collected from control and dose group animals at the day of sacrifice, were below 0.05 µg Co, below 5.23 µg Al and below 15.64 µg Zn/L plasma.

It is concluded that the pigment was well tolerated and that no signs of systemic toxicity whatsoever were observed had been seen in rats when administered at a dose of 1000 mg/kg bw/day for up to 28 days.Either no or only marginal increases in Al, Zn and Co plasma concentrations were observed, and only a minor fraction (<0.003%) of the total administered dose of Co, Al and Zn was collected via urine, documenting the lack of bioavailability of this pigment. The no observed adverse effect level (NOAEL) in rats is 1000 mg/kg/day.

1: Aluminium is excreted primarily via urine, with less than 0.1% via bile (Lote & Saunders (1991): Clin. Science 81,289-295). The majority of systemically available cobalt is excreted rapidly via urine (Nordberg et al. (2014): Handbook on the toxicology of metals, Academ. Press, New York).

 

Animal data - inhalation route:

In this 90-day repeated dose toxicity by inhalation study, rats were exposed via nose-only inhalation towards aerosol concentrations of 0.4, 1.5 and 6 mg/m³ of Cobalt Zinc Aluminate Blue Spinel.

All animals survived the test period and were euthanized at scheduled dates. Effects indicating systemic toxicity were not observed. Sex-specific differences were not detected.

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

Some adaptive exposure-related findings were detected in the investigated high dose group (Pigment 1 - Cobalt Zinc Aluminate Blue Spinel) within the lung, the nasal cavity, and the lung-associated lymph nodes: accumulation of particle-laden macrophages. Very slight multifocal hyaline droplets were seen in the nasal epithelium. These lesions are considered to represent a non-adverse adaptive change. Moreover, very slight multifocal granulocytic cell infiltration in the alveoli were observed. A single male showed very slight bronchiolo-alveolar hyperplasia of the bronchial type (bronchiolization).

Under the conditions of this test, based on very slight granulocytic cell infiltration in the lung, a NOAEC of 6 mg/m³ was derived for both sexes, being the maximum tolerated concentration based on the more than 2-fold increase lung clearance half-time (further details are reported in the study record in the toxicokinetics section). Consequently, the pigment is to be regarded as inert material when tested up to the maximum tolerated concentration as defined by significant impairment of lung clearance (Driscoll and Borm, 2020)*.

 

 

*References:

Driscoll, K.E. and Borm, P.J.A., 2020. Expert workshop on the hazards and risks of poorly soluble low toxicity particles. Inhal Toxicol 32(2):53-62.

Justification for classification or non-classification

No signs of systemic toxicity whatsoever were observed in rats when administered at a dose of 1000 mg/kg bw/day for up to 28 days and after 90 -days nose-only inhalation up to the maximum tolerated concentration.

No classification for Specific Target Organ Toxicity-Repeated Exposure (STOT-RE) according to EC Regulation No. 1272/2008 is required.